Useful polypeptides

ABSTRACT

The present invention provides a novel polypeptide having a β1,3-N-acetylglucosaminyltransferase activity; a method for producing the polypeptide; a DNA which encodes the polypeptide; a recombinant vector into which the DNA is inserted; a transformant comprising the recombinant vector; a method for producing a sugar chain or complex carbohydrate, using the polypeptide; a method for producing a sugar chain or complex carbohydrate, using the transformant; an antibody which recognizes the polypeptide; a method for screening a substance which changes the expression of the gene which encodes the polypeptide; and a method for screening a substance which changes the activity of the polypeptide.

TECHNICAL FIELD

The present invention relates to a novel polypeptide having aβ1,3-N-acetylglucosaminyltransferase activity; a sugar chainsynthesizing agent comprising the polypeptide as an active ingredient; aDNA which encodes the polypeptide; an agent for detecting inflammation,cancer or metastasis, comprising the DNA; a recombinant DNA which isobtained by inserting the DNA into a vector; a transformant carrying therecombinant DNA; a method for producing the polypeptide by using thetransformant; a method for producing a sugar chain or complexcarbohydrate by using the polypeptide; a process for producing a sugarchain or complex carbohydrate by using the transformant; a method fordetecting inflammation, cancer or metastasis by using an oligonucleotideobtained from the DNA encoding the polypeptide; an antibody whichrecognizes the polypeptide; an immunohistostaining method by using theantibody; an immunohistostaining agent comprising the antibody; an agentfor diagnosing inflammation, cancer or metastasis; a method forscreening a compound capable of changing theβ1,3-N-acetylglucosaminyltransferase activity of the polypeptide; amethod for screening a compound capable of changing the expression ofthe corresponding gene; a promoter DNA capable of controlling thetranscription of the corresponding gene; a method for screening acompound capable of changing the efficiency of transcription by thepromoter DNA; a compound obtained by these screening methods; a knockout animal in which the corresponding gene is deleted or mutated; andthe like.

BACKGROUND ART

It is considered that sugar chains are related to vital phenomena suchas development, differentiation and cell recognition and also deeplyrelated to the development and progress of inflammations, cancers,autoimmune diseases and a large number of other diseases [Fukuda, M.,Cell Surface Carbohydrates and Cell Development, CRC Press, Bosa Raton,Fla. (1992), Glycobiology, 3, 97 (1993)].

Sugar chains are present not only as glycoproteins, proteoglycans orglycolipids by addition to proteins or lipids and but also asoligosaccharides.

Gal β1,3-N-acetylglucosaminyltransferase is an enzyme having an activityof transferring N-acetylglucosamine to galactose residues present at thenon-reducing terminal of a sugar chain via a β1,3-linkage, and involvedin the synthesis of a sugar chain having a GlcNAc β1-3Gal structure.Sugar chains having a GlcNAcβ1-3Gal structure are present inN-glycosylated sugar chains and O-glycosylated sugar chains ofglycoproteins and also in neolacto-series and lacto-series glycolipids,and in oligosaccharides.

With regard to a Gal β1,3-N-acetylglucosaminyltransferase, its partialpurification has so far been reported [J. Biol. Chem., 268, 27118(1993), J. Biol. Chem., 267, 2994 (1992), J. Biol. Chem., 263, 12461(1988), Jpn. J. Med. Sci. Biol., 42, 77 (1989)]. Also, two types of Galβ1,3-N-acetylglucosaminyltransferase have been cloned [Proc. Natl. Acad.Sci. USA., 94, 14294 (1997), Proc. Natl., Acad. Sci. USA., 96, 406(1999)]. The presence of other types of Galβ1,3-N-acetylglucosaminyltransferases has not been clarified.

Since sugar chains having a GlcNAcβ1-3Gal structure are present in greatnumbers, it is considered that two or more enzymes having differentreceptor substrate specificity or expressing at different tissue arepresent as Gal β1,3-N-acetylglucosaminyltransferases, and they arerespectively taking part in different functions. Thus, it is animportant subject to clone a Gal β1,3-N-acetylglucosaminyltransferasedifferent from the two enzymes so far cloned, to examine its receptorsubstrate specificity as well as expression and distribution, and toelucidate its relation to biological functions and diseases.

It is known that lacto-N-neotetraose (Galβ1-4GlcNAcβ1-3Galβ1-4Glc) andlacto-N-tetraose (Galβ1-3GlcNAcβ1-3Galβ1-4Glc) or variousoligosaccharides containing them as the core structure are present inhuman milk [Acta Paediatrica, 82, 903 (1993)]. It is considered thatthese oligosaccharides have a function of preventing babies from viraland microbial infections and a function of neutralizing toxins. Also,they have an activity of accelerating growth of Bifidobacterium that isa good enteric bacterium.

It will be markedly useful from the industrial point of view if theseoligosaccharides contained in human milk or milk containing them can beefficiently produced. If a gene for a Galβ1,3-N-acetylglucosaminyltransferase involved in the synthesis of theseoligosaccharides contained in human milk is obtained, theseoligosaccharides would be efficiently synthesized, but such an enzymehas not been found.

Among a large number of sugar chains having a GlcNAcβ1-3Gal structure,particularly poly-N-acetyllactosamine sugar chains serve as core sugarchains of many functional sugar chains (selectin ligand sugar chains,microbial or viral receptor sugar chains, SSEA-1 sugar chains,cancer-related sugar chains and the like) and deeply relate to diseases,such as embryogenesis, cell differentiation or diseases such asinflammation, cancer and the like.

Since there is a possibility that Galβ1,3-N-acetylglucosaminyltransferases involved in the synthesis ofpoly-N-acetyllactosamine sugar chains functioning in respective casesare different, it is an important subject to clone a Galβ1,3-N-acetylglucosaminyltransferase which is different from the twoenzymes so far cloned and to estimate functions of respective enzymesbased on their receptor substrate specificity, expression distributionand the like.

The poly-N-acetyllactosamine sugar chain is synthesized by the alternatefunctions of a GlcNAc β1,4-N-galactosyltransferase activity and a Galβ1,3-N-acetylglucosaminyltransferase activity. Regardingβ1,4-galactosyltransferases, genes for 4 enzymes (β4Gal-T1, β4Gal-T2,β4Gal-T3 and β4Gal-T4) have been cloned, and the receptor substratespecificity of each enzyme has been analyzed [J. Biol. Chem., 272, 31979(1997), J. Biol. Chem., 273, 29331 (1997)].

Accordingly, a poly-N-acetyllactosamine sugar chain can be synthesizedin vitro by using a GlcNAc β1,4-galactosyltransferase and a Galβ1,3-N-acetylglucosaminyltransferase. Also, a poly-N-acetyllactosaminesugar chain or a complex carbohydrate to which the sugar chain is addedcan be synthesized by co-expressing a GlcNAc β1,4-galactosyltransferasegene and a Gal β1,3-N-acetylglucosaminyltransferase gene in cells.

Since a GlcNAc β1,4-galactosyltransferase is expressed in almost allcells, poly-N-acetyllactosamine sugar chain or a sugar to which thesugar chain is added can be synthesized by expressing a Galβ1,3-N-acetylglucosaminyltransferase gene in cells.

It is known that a poly-N-acetyllactosamine sugar chain is morefrequently expressed in cancer cells in comparison with correspondingnormal cells [J. Biol. Chem., 259, 10834 (1984), J. Biol. Chem., 261,10772 (1986), J. Biol. Chem., 266, 1772 (1991), J. Biol. Chem., 267,5700 (1992)].

It is expected that a poly-N-acetyllactosamine sugar chain having sialylLewis x sugar chain might be a medicament having an anti-inflammatoryeffect or a metastasis inhibiting effect, as a selectin antagonist.

A partially purified β1,3-N-acetylglucosaminyltransferase has been usedin synthesizing the poly-N-acetyllactosamine sugar chain moiety of theseoligosaccharides. But since supply of this enzyme is a rate-limitingstep, it is difficult to synthesize the poly-N-acetyllactosamine sugarchain in a large amount [Glycobiology, 7, 453 (1997)].

On the other hand, a poly-N-acetyllactosamine sugar chain can also besynthesized by chemical synthesis, but its synthesis requiresconsiderably complex steps [Tetrahedron Letter, 24, 5223 (1997)].

Thus, an efficient process for synthesizing a poly-N-acetyllactosaminesugar chain is in demand. Although two types of Galβ1,3-N-acetylglucosaminyltransferase so far cloned and genes for theseenzymes can be used, it is considered that the use of other Galβ1,3-N-acetylglucosaminyltransferases having different substratespecificity and different function or their genes may be efficient insome cases depending on the purpose.

Since a poly-N-acetyllactosamine sugar chain contributes to thestabilization of protein [J. Biol. Chem., 265, 20476 (1990)], it isconsidered that a protein can be stabilized by artificially adding apoly-N-acetyllactosamine sugar chain to a desired protein. Also, since aclearance rate of proteins in blood from the kidney becomes slower asthe effective molecular weight of the protein becomes larger, it isconsidered that the clearance rate from the kidney can be reduced andblood stability can be increased by artificially adding apoly-N-acetyllactosamine sugar chain to a desired protein to therebyincrease the size of the effective molecular weight. In addition, thereis a possibility that a desired protein can be targeted to a specifiedcell. Cases in which synthesizing ability of a poly-N-acetyllactosaminesugar chain has been increased are shown below.

It is shown that a poly-N-acetyllactosamine sugar chain is added tosugar chains of a membrane-bound glycoprotein of cells when F9 cell istreated with retinoic acid or Swiss 3T3 cell is treated with TGF-β [J.Biol. Chem., 268, 1242 (1993), Biochim. Biophys. Acta., 1221, 330(1994)].

It is shown that, when N-ras proto-oncogene is expressed in NIH3T3 cell,activities of β1,4-galactosyltransferase andβ1,3-N-acetylglucosaminyltransferase which are involved in the synthesisof poly-N-acetyllactosamine sugar chains are increased and the amount ofpoly-N-acetyllactosamine sugar chains in the N-linked sugar chain of amembrane protein is increased [J. Biol. Chem., 266, 21674 (1991)].

When the gene for core 2 β1,6-N-acetylglucosaminyltransferase isexpressed in T-cell line EL-4, the molecular weight of a cell surfacemembrane protein CD43, CD45 or CD44 is increased [J. Biol. Chem., 271,18732 (1996)]. The phenomenon is considered to be due to that the sugarchain synthesized by the core 2 β1,6-N-acetylglucosaminyltransferasebecomes a good substrate of a β1,3-N-acetylglucosaminyltransferase whichis involved in the synthesis of a oly-N-acetyllactosamine sugar chain.

Also, it is known that when HL-60 cell is cultured at 27° C., the amountof poly-N-acetyllactosamine sugar chains added to lamp-1 or lamp-2 isincreased [J. Biol. Chem., 266, 23185 (1991)].

However, there are no reports on the efficient production of recombinantglycoproteins to which a poly-N-acetyllactosamine sugar chain is added,in host cells suitable for the production of recombinant glycoproteins(e.g., Namalwa cell, Namalwa KJM-1 cell, CHO cell and the like).Accordingly, development of a process for producing a recombinantglycoprotein to which a poly-N-acetyllactosamine sugar chain is added isan industrially important subject.

When mechanisms of inflammatory reaction and metastasis are taken intoconsideration, it is expected that inflammatory reaction can beinhibited and metastasis can be prevented by inhibiting expression ofpoly-N-acetyllactosamine sugar chain on leukocytes and cancer cells. Ifa gene for a Gal β1,3-N-acetylglucosaminyltransferase which is involvedin the synthesis of a poly-N-acetyllactosamine sugar chain on leukocytesand cancer cells can be obtained, there is a possibility that expressionof the poly-N-acetyllactosamine sugar chain on leukocytes and cancercells can be inhibited by inhibiting expression of the gene.

Also, if a gene for a Gal β1,3-N-acetylglucosaminyltransferase which isinvolved in the synthesis of a poly-N-acetyllactosamine sugar chain onleukocytes and cancer cells can be obtained, there is a possibility thatinflammatory diseases and the malignancy of cancers can be diagnosed byexamining the expression level of the gene or examining the expressionlevel of a protein encoded by the gene.

Since it is considered that Gal β1,3-N-acetylglucosaminyltransferasesinvolved in the synthesis of a poly-N-acetyllactosamine sugar chain onspecified leukocytes and cancer cells are different, it is necessary toclone and examine an enzyme different from the enzymes so far cloned.

Each of Gal β1,3-N-acetylglucosaminyltransferases expressed in a cell ortissue in which two or more Gal β1,3-N-acetylglucosaminyltransferasesare expressed cannot be specified, and enzymological characteristics ofeach of the Gal β1,3-N-acetylglucosaminyltransferases cannot beclarified by enzymological analyses in which extracts of cells ortissues are used as enzyme sources.

In order to detect the expression of a specified Galβ1,3-N-acetylglucosaminyltransferase, it is necessary to use animmunological detection method by using a specific antibody or adetection method based on the nucleotide sequence of the gene (e.g.,Northern hybridization or PCR). Accordingly, it is necessary to clone aGal β1,3-N-acetylglucosaminyltransferase different from the enzymes sofar cloned and compare their expressions.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide medicaments foranti-inflammatory, anti-infectious, metastasis-inhibiting and the like,foods such as dairy products and the like, a method for improvingprotein, and a method for diagnosing inflammatory diseases andmalignancy of cancers by using a novel polypeptide having a Galβ1,3-N-acetylglucosaminyltransferase activity.

The present invention relates to the following subject matters (1) to(62).

(1) A sugar chain synthesizing agent, which comprises, as an activeingredient, a polypeptide selected from the group consisting of thefollowing (a), (b), (c), (d), (e), (f), (g) and (h), and having anactivity involved in synthesis of a poly-N-acetyllactosamine sugarchain:

-   -   (a) a polypeptide comprising the amino acid sequence represented        by SEQ ID NO:1,    -   (b) a polypeptide comprising an amino “acid sequence of        positions 41-397 in the amino acid sequence represented by SEQ        ID NO:1,    -   (c) a polypeptide comprising the amino acid sequence represented        by SEQ ID NO:2,    -   (d) a polypeptide comprising an amino acid sequence of positions        45-372 in the amino acid sequence represented by SEQ ID NO:2,    -   (e) a polypeptide comprising the amino acid sequence represented        by SEQ ID NO:3,    -   (f) a polypeptide comprising an amino acid sequence of positions        45-372 in the amino acid sequence represented by SEQ ID NO:3,    -   (g) a polypeptide comprising the amino acid sequence represented        by SEQ ID NO:4, and    -   (h) a polypeptide comprising an amino acid sequence of positions        62-378 in the amino acid sequence represented by SEQ ID NO:4.        (2) The sugar chain synthesizing agent according to (1), wherein        the polypeptide comprises an amino acid sequence in which one or        more amino acids are deleted, substituted or added in the amino        acid sequence of the polypeptide according to (1), and has an        activity involved in the synthesis of a poly-N-acetyllactosamine        sugar chain.        (3) The sugar chain synthesizing agent according to (1) or (2),        wherein the activity involved in the synthesis of a        poly-N-acetyllactosamine sugar chain is a        β1,3-N-adetylglucosaminyltransferase activity.        (4) A polypeptide which is selected from the group consisting of        the following (a), (b), (c) and (d):    -   (a) a polypeptide comprising the amino acid sequence represented        by SEQ ID NO:3,    -   (b) a polypeptide comprising an amino acid sequence of positions        45-372 in the amino acid sequence represented by SEQ ID NO:3,    -   (c) a polypeptide comprising the amino acid sequence represented        by SEQ ID NO:4, and    -   (d) a polypeptide comprising an amino acid sequence of positions        62-378 in the amino acid sequence represented by SEQ ID NO:4.        (5) A polypeptide comprising an amino acid sequence in which one        or more amino acids are deleted, substituted or added in the        amino acid sequence represented by SEQ ID NO:4, and having an        activity involved in the synthesis of a poly-N-acetyllactosamine        sugar chain.        (6) A polypeptide which is the following (a) or (b), and has an        activity involved in the synthesis of a poly-N-acetyllactosamine        sugar chain;    -   (a) a polypeptide which comprises an amino acid sequence of        positions 41-397 in the amino acid sequence represented by SEQ        ID NO:1 and is free of an amino acid sequence of positions 1-33        in the amino acid sequence represented by SEQ ID NO:1, or    -   (b) a polypeptide which comprises an amino acid sequence of        positions 45-372 in the amino acid sequence represented by SEQ        ID NO:2 and is free of the amino acid sequence represented by        SEQ ID NO:2.        (7) A polypeptide comprising an amino acid sequence in which one        or more amino acids are deleted, substituted or added in the        amino acid sequence of the polypeptide according to (6), and        having an activity involved in the synthesis of a        poly-N-acetyllactosamine sugar chain.        (8) The polypeptide according to any one of (4) to (7), wherein        the activity involved in the synthesis of a        poly-N-acetyllactosamine sugar chain is a        β1,3-N-acetylglucosaminyltransferase activity.        (9) The polypeptide according to (8), wherein the        β1,3-N-acetylglucosaminyltransferase activity of the polypeptide        is an activity of transferring N-acetylglucosamine to a        galactose residue present in the non-reducing terminal of a        sugar chain via a β1,3-linkage.        (10) The polypeptide according to (8) or (9), wherein the        β1,3-N-acetylglucosaminyltransferase activity is an activity of        transferring N-acetylglucosamine via a β1,3-linkage to a        galactose residue present in the non-reducing terminal of an        acceptor substrate selected from i) N-adetyllactosamine        (Galβ1-4GlcNAc) or lactose (Galβ1-4Glc), ii) an oligosaccharide        having an N-acetyllactosamine or lactose structure at the        non-reducing terminal, and iii) a complex carbohydrate having an        N-acetyllactosamine or lactose structure at the non-reducing        terminal.        (11) A glycosyltransferase which is a polypeptide selected from        the group consisting of the following (a), (b), (c), (d), (e),        (f), (g) and (h), and has an activity involved in the synthesis        of a poly-N-acetyllactosamine sugar chain;    -   (a) a polypeptide comprising the amino acid sequence represented        by SEQ ID NO:1,    -   (b) a polypeptide comprising an amino acid sequence of positions        41-397 in the amino acid sequence represented by SEQ ID NO:1,    -   (c) a polypeptide comprising the amino acid sequence represented        by SEQ ID NO:2,    -   (d) a polypeptide comprising an amino acid sequence of positions        45-372 in the amino acid sequence represented by SEQ ID NO:2,    -   (e) a polypeptide comprising the amino acid sequence represented        by SEQ ID NO:3,    -   (f) a polypeptide comprising an amino acid sequence of positions        45-372 in the amino acid sequence represented by SEQ ID NO:3,    -   (g) a polypeptide comprising the amino acid sequence represented        by SEQ ID NO:4, and    -   (h) a polypeptide comprising an amino acid sequence of positions        62-378 in the amino acid sequence represented by SEQ ID NO:4.        (12) The glycosyltransferase according to (11), wherein the        polypeptide which comprises an amino acid sequence in which one        or more amino acids are deleted, substituted or added in the        amino acid sequence of the polypeptide according to (11), and        has an activity involved in the synthesis of a        poly-N-acetyllactosamine sugar chain.        (13) A DNA which encodes the polypeptide according to any one        of (4) to (10).        (14) A DNA which comprises the nucleotide sequence represented        by SEQ ID NO:7 or 8.        (15) A DNA which hybridizes with the DNA comprising the        nucleotide sequence represented by SEQ ID NO:8 under stringent        conditions and encodes a polypeptide having a        β1,3-N-acetylglucosaminyltransferase activity.        (16) An agent for detecting inflammation, cancer or metastasis,        which comprises the DNA according to any one of (13) to (15).        (17) A recombinant DNA which is obtained by inserting the DNA        according to any one of (13) to (15) into a vector.        (18) The recombinant DNA according to (17), wherein the        recombinant DNA is a plasmid selected from the group consisting        of plasmids pAMo-G4-2, pAMo-G7, pAMoF2-G4, pVL1393-F2G4,        pBS-G4-2, and pT7B-G7.        (19) A transformant which comprises the recombinant DNA        according to (17) or (18).        (20) The transformant according to (19), wherein the        transformant is selected from the group consisting of a        microorganism, an animal cell, a plant cell, an insect cell, a        non-human transgenic animal, and a transgenic plant.        (21) The transformant according to (20), wherein the        microorganism belongs to the genus Escherichia.        (22) A biologically pure culture of Escherichia coli        MM294/pBS-G3 (FERM BP-6694), Escherichia coil MM294/pBS-G4 (FERM        BP-6695), or Escherichia coli MM294/pT7B-G7 (FERM BP-6696).        (23) The transformant according to (20), wherein the animal cell        is selected from the group consisting of a mouse myeloma cell, a        rat myeloma cell, a mouse hybridoma cell, CHO cell, BHK cell,        African green monkey kidney cell, Namalwa cell, Namalwa KJM-1        cell, a human fetal kidney cell, and a human leukemia cell.        (24) The transformant according to (20), wherein the insect        cell, is selected from the group consisting of a Spodoptera        frugiperda ovary cell, a Trichoplusia ni ovary cell, and a        Bombyx mori ovary cell.        (25) A process for producing the polypeptide according to any        one of (4) to (10), which comprises:    -   culturing a transformant carrying a recombinant DNA obtained by        inserting a DNA encoding the polypeptide into a vector in a        medium to produce and accumulate the polypeptide in the culture;        and    -   recovering the polypeptide from the culture.        (26) The process according to (25), wherein the transformant is        selected from the group consisting of a microorganism, an animal        cell, a plant cell, and an insect cell.        (27) A process for producing the polypeptide according to any        one of (4) to (10), which comprises:    -   rearing a non-human transgenic animal carrying a recombinant DNA        obtained by inserting a DNA encoding the polypeptide into a        vector to produce and accumulate the polypeptide in the animal;        and    -   recovering the polypeptide from the animal.        (28) The process according to (27), wherein said prodution and        accumulation are carried out in milk of the animal.        (29) A process for producing the polypeptide according to any        one of (4) to (10), which comprises:    -   cultivating a transgenic plant carrying a recombinant DNA        obtained by inserting a DNA encoding the polypeptide into a        vector to produce; and accumulating the polypeptide in the        plant; and    -   recovering the polypeptide from the plant.        (30) A process for producing the polypeptide according to any        one of (4) to (10), wherein the polypeptide is synthesized by an        in vitro transcription translation system using a DNA encoding        the polypeptide.        (31) A process for producing a sugar chain or complex        carbohydrate, which comprises:    -   selecting, as an enzyme source, the sugar chain synthesizing        agent according to (1) or (2);    -   allowing    -   (a) the enzyme source,    -   (b) an acceptor substrate selected from i) N-acetyllactosamine        (Galβ1-4GlcNAc), Galβ1-3GlcNAc or lactose (Galβ1-4Glc), ii) an        oligosaccharide having an N-acetyllactosamine, Galβ1-3GlcNAc or        lactose structure at the non-reducing end, and iii) a complex        carbohydrate having an N-acetyllactosamine, Galβ1-3GlcNAc or        lactose structure at the non-reducing terminal, and    -   (c) uridine-5′-diphosphate N-acetylglucosamine to be present in        an aqueous medium to produce and accumulate a sugar chain or        complex carbohydrate in which N-acetylglucosamine is added to a        galactose residue of the acceptor substrate via a β1,3-linkage;        and    -   recovering the sugar chain or complex carbohydrate from the        aqueous medium.        (32) A process for producing a sugar chain or complex        carbohydrate to which galactose is added, which comprises:    -   selecting, as an acceptor substrate, the        N-acetylglucosamine-added reaction product obtained by the        method according to (31);    -   allowing    -   (a) the acceptor substrate,    -   (b) a GlcNAc β1,4-galactosyltransferase, and    -   (c) uridine-5′-diphosphogalactose are allowed to be present in        an aqueous medium to produce and accumulate a sugar chain or        complex carbohydrate in which galactose is added to        N-acetylglucosamine residue at the non-reducing terminal of the        acceptor substrate via a β1,4-linkage; and    -   recovering the galactose-added sugar chain or complex        carbohydrate from the aqueous medium.        (33) A process for producing a sugar chain or complex        carbohydrate to which a poly-N-acetyllactosamine sugar chain is        added, which comprises:    -   selecting, as an enzyme source, the sugar chain synthesizing        agent according to (1) or (2);    -   allowing    -   (a) the enzyme source,    -   (b) a GlcNAc β1,4-galactosyltransferase,    -   (c) a acceptor substrate selected from i) N-acetyllactosamine        (Galβ1-4GlcNAc), Galβ1-3GlcNAc or lactose (Galβ1-4Glc), ii) an        oligosaccharide having an N-acetyllactosamine, Galβ1-3GlcNAc or        a lactose structure at the non-reducing end, iii) a complex        carbohydrate having an N-acetyllactosamine, Galβ1-3GlcNAc or a        lactose structure at the non-reducing terminal, and iv) the        reaction product obtained by the process according to (31) or        (32),    -   (d) uridine-5′-diphospho-N-acetylglucosamine, and    -   (e) uridine-5′-diphosphogalactose to be present in an aqueous        medium to produce and accumulate a sugar chain or complex        carbohydrate in which a poly-N-acetyllactosamine sugar chain is        added to the non-reducing terminal of the acceptor substrate;    -   recovering the poly-N-acetyllactosamine sugar chain-added sugar        chain or complex carbohydrate from the aqueous medium.        (34) A process for producing a sugar chain or complex        carbohydrate, which comprises:    -   culturing a transformant carrying a recombinant DNA obtained by        inserting a DNA encoding a polypeptide which is the active        ingredient of the sugar chain synthesizing agent according        to (1) or (2) into a vector in a medium to produce and        accumulate a sugar chain comprising a saccharide selected from        the group consisting of a saccharide having a        GlcNAcβ1-3Galβ1-4GlcNAc structure, a saccharide having a        GlcNAcβ1-3Galβ1-3GlcNAc structure, a saccharide having a        GlcNAcβ1-3Galβ1-4Glc structure, a saccharide having a        (Galβ1-4GlcNAcβ1-3)_(n)Galβ1-4GlcNAc structure wherein n is 1 or        more, and a saccharide having a        (Galβ1-4GlcNAcβ1-3)_(n)Galβ1-4Glc structure wherein n is 1 or        more, or a complex carbohydrate comprising the sugar chain, in        the culture; and    -   recovering the sugar chain or complex carbohydrate from the        culture.        (35) The process according to (34), wherein the transformant is        a microorganism, an animal cell, a plant cell or an insect cell.        (36) A process for producing a sugar chain or complex        carbohydrate, which comprises:    -   rearing a non-human transgenic animal carrying a recombinant DNA        obtained by inserting a DNA encoding a polypeptide which is the        active ingredient of the sugar chain synthesizing agent        according to (1) or (2) into a vector to produce and accumulate        a sugar chain comprising a saccharide selected from the group        consisting of a saccharide having a GlcNAcβ1-3Galβ1-4GlcNAc        structure, a saccharide having a GlcNAcβ1-3Galβ1-3GlcNAc        structure, a saccharide having a GlcNAcβ1-3Galβ1-4Glc structure,        a saccharide having a (Galβ1-4GlcNAcβ1-3)_(n)Galβ1-4GlcNAc        structure wherein n is 1 or more, and a saccharide having a        (Galβ1-4GlcNAcβ1-3)_(n)Galβ1-4Glc structure wherein n is 1 or        more, or a complex carbohydrate comprising the sugar chain, in        the animal; and    -   recovering the sugar chain or complex carbohydrate from the        animal.        (37) A process for producing a sugar chain or complex        carbohydrate, which comprises:    -   cultivating a transgenic plant carrying a recombinant DNA        obtained by inserting a DNA encoding a polypeptide which is the        active ingredient of the sugar chain synthesizing agent        according to (1) or (2) into a vector to produce and accumulated        a sugar chain comprising a saccharide selected from the group        consisting of a saccharide having a GlcNAcβ1-3Galβ1-4GlcNAc        structure, a saccharide having a GlcNAcβ1-3Galβ1-3GlcNAc        structure, a saccharide having a GlcNAcβ1-3Galβ1-4Glc structure,        a saccharide having a (Galβ1-4GlcNAcβ1-3)_(n)Galβ1-4GlcNAc        structure wherein n is 1 or more, and a saccharide having a        (Galβ1-4GlcNAcβ1-3)_(n)Galβ1-4Glc structure wherein n is 1 or        more, or a complex carbohydrate comprising the sugar chain, in        the plant; and    -   recovering the sugar chain or complex carbohydrate from the        plant.        (38) The process according to any one of (31) to (37), wherein        the complex carbohydrate is a complex carbohydrate selected from        a glycoprotein, a glycolipid, a proteoglycan, a glycopeptide, a        lipopolysaccharide, a peptidoglycan, and a glycoside in which a        sugar chain is bound to a steroid compound.        (39) The process according to (36), wherein said production and        accumulation are carried out in milk of the animal.        (40) A method for determining an expression level of a gene        encoding a polypeptide having the amino acid sequence        represented by any one of SEQ ID NO:1 to 4, which comprises        carrying out hybridization by using the DNA according to any one        of (13) to (15).        (41) An oligonucleotide which is selected from an        oligonucleotide having a sequence identical to continuous 6 to        60 nucleotides of a DNA having the nucleotide sequence        represented by SEQ ID NO:8, an oligonucleotide having a sequence        complementary to the oligonucleotide, and a derivative of the        oligonucleotide.        (42) The oligonucleotide according to (41), wherein the        derivative of oligonucleotide is selected from an        oligonucleotide derivative in which a phosphodiester bond in the        oligonucleotide is converted into a phosphorothioate bond, an        oligonucleotide derivative in which a phosphodiester bond in the        oligonucleotide is converted into an N3′-P5′ phosphoamidate        bond, an oligonucleotide derivative in which ribose and a        phosphodiester bond in the oligonucleotide are converted into a        peptide-nucleic acid bond, an oligonucleotide derivative in        which uracil in the oligonucleotide is substituted with C-5        propynyluracil, an oligonucleotide derivative in which uracil in        the oligonucleotide is substituted with C-5 thiazoleuracil, an        oligonucleotide derivative in which cytosine in the        oligonucleotide is substituted with C-5 propynylcytosine, an        oligonucleotide derivative in which cytosine in the        oligonucleotide is substituted with phenoxazine-modified        cytosine, an oligonucleotide derivative in which ribose in the        oligonucleotide is substituted with 2′-O-propylribose, and an        oligonucleotide derivative in which ribose in the        oligonucleotide is substituted with 2′-methoxyethoxyribose.        (43) A method for determining an expression level of a gene        encoding the polypeptide according to any one of (4) to (10),        which comprises carrying out a polymerase chain reaction by        using an oligonucleotide selected from an oligonucleotide having        a sequence identical to continuous 6 to 60 nucleotides of a        nucleotide sequence of a DNA encoding the polypeptide according        to any one of (4) to (10), an oligonucleotide having a sequence        complementary to the oligonucleotide, and a derivative of the        oligonucleotide.        (44) A method for detecting inflammation, cancer or metastasis,        which comprises using the method according to (40) or (43).        (45) A method for inhibiting transcription of a DNA encoding the        polypeptide according to any one of (4) to (10) or translation        of the mRNA corresponding to the DNA, which comprises using an        oligonucleotide selected from an oligonucleotide having a        sequence identical to continuous 6 to 60 nucleotides of a        nucleotide sequence of the DNA according to any one of (13) to        (15), an oligonucleotide having a sequence complementary to the        oligonucleotide, and a derivative of the oligonucleotide.        (46) An oligonucleotide selected from an oligonucleotide having        a sequence identical to continuous 6 to 60 nucleotides of a        nucleotide sequence of a DNA encoding the polypeptide according        to any one of (4) to (10), an oligonucleotide having a sequence        complementary to the oligonucleotide, and a derivative of the        oligonucleotide.        (47) An antibody which recognizes the polypeptide according to        any one of (4) to (10).        (48) A method for immunologically detecting the polypeptide        according to any one of (4) to (10), which comprises using the        antibody according to (47).        (49) An immunohistostaining method, which comprises detecting        the polypeptide according to any one of (4) to (10) by using the        antibody according to (47).        (50) An immunohistostaining agent, comprising the antibody        according to (47).        (51) An agent for diagnosing inflammation, cancer or metastasis,        which comprises the antibody according to (47).        (52) A method for screening a compound capable of changing the        β1,3-N-acetylglucosaminyltransferase activity of the polypeptide        according to any one of (4) to (10), which comprises bringing        the polypeptide into contact with a test sample.        (53) A method for screening a compound capable of changing the        expression of a gene encoding the polypeptide according to any        one of (4) to (10), which comprises:    -   bringing a cell in which the polypeptide is expresssed, into        contact with a test sample; and    -   measuring the content of the poly-N-acetyllactosamine sugar        chain by using an antibody or a lectin capable of recognizing        the poly-N-acetyllactosamine sugar chain.        (54) A method for screening a compound which changes the        expression of a gene encoding the polypeptide according to any        one of (4) to (10), which comprises:    -   bringing a cell expressing the polypeptide into contact with a        test sample; and    -   measuring the polypeptide content by using the antibody        according to (47).        (55) A promoter DNA, which is capable of controlling the        transcription of the gene encoding the polypeptide according to        any one of (4) to (10).        (56) The promoter DNA according to (55), wherein the promoter        DNA which functions in a cell selected from a leukocyte, a small        intestine cell, a large intestine cell, a spleen cell, a stomach        cell, a large bowel cancer cell, a pancreatic cancer cell, and a        gastric cancer cell.        (57) The promoter DNA according to (55) or (56), wherein the        promoter DNA which derives from human or mouse.        (58) A method for screening a compound capable of changing the        efficiency of transcription by the promoter according to any one        of (55) to (57), which comprises:    -   transforming an animal cell with a plasmid containing the        promoter DNA and a reporter gene linked to downstream of the        promoter DNA;    -   bringing the resulting transformant into contact with a test        sample; and    -   measuring the content of the translation product of the reporter        gene.        (59) The method according to (58), wherein the reporter gene is        selected from a chloramphenicol acetyltransferase gene, a        β-galactosidase gene, a β-lactamase gene, a luciferase gene, and        a green fluorescent protein gene.        (60) A compound obtained by the method according to any one        of (52) to (54), (58) and (59).        (61) A knock out non-human animal in which a DNA encoding the        polypeptide according to any one of (4) to (10) is deleted or        mutated.        (62) The knock out non-human animal according to (61), wherein        the knock out non-human animal is a mouse.

The present invention is explained below in detail.

(1) Acquisition of a DNA Encoding a Protein Homologous to a GlcNAcβ1,3-galactosyltransferase (β3Gal-T1) and Production of the DNA and theOligonucleotide

β3Gal-T1 (another name WM1) is a GlcNAc β1,3-galactosyltransferaseinvolved in the synthesis of a Galβ1-3GlcNAc structure (JapanesePublished Unexamined Patent Application No. 181759/94). A gene havinghomology with the gene encoding this enzyme or a gene having apossibility of encoding a protein having homology with this enzyme atthe amino acid level is searched based on gene data bases by using aprogram such as Blast [Altschul et al., J. Mol. Biol., 215, 403(1990)],” FrameSearch method (manufactured by Compugen) or the like. Asdata bases, a public data base such as GenBank or the like can be used,and a private data base can also be used. The presence of DNAs havingthe corresponding sequence can be detected by carrying out polymerasechain reaction (hereinafter referred to as “PCR”) [Molecular Cloning, ALaboratory Manual, 2nd Ed., Cold Harbor Laboratory Press (1989)(hereinafter referred to as “Molecular Cloning, 2nd Ed.”) and PCRProtocols, Academic Press (1990)] by using a single-stranded cDNA or acDNA library prepared from various organs or various cells as a templateand using primers specific for the corresponding sequence. Also, a DNAfragment having the corresponding sequence can be obtained in the samemanner.

When the thus obtained DNA fragment is not a full length, itsfull-length cDNA can be obtained as follows.

A full-length cDNA can be obtained by screening an organ- orcell-derived cDNA library in which the presence of the DNA has beenconfirmed, by using the DNA fragment obtained in the above as a probe.

Also, a 5′-end side fragment and a 3′-end side fragment of a cDNA havingthe corresponding sequence can be obtained by carrying out 5′-RACEmethod and 3′-RACE method using a single-stranded cDNA or cDNA libraryas the template in which the presence of the DNA has been confirmed. Byligating both fragments, a full-length cDNA can be obtained.

The single-stranded cDNA derived from various organs or various cellscan be prepared in accordance with the usual method or by using acommercially available kit. An example is shown below.

A total RNA is extracted from various organs or various cell by the acidguanidium thiocyanate phenol-chloroform method [Anal. Biochem., 162, 156(1987)]. If necessary, the total RNA is treated with deoxyribonuclease I(manufactured by Life Technologies) to degrade possible chromosomal DNAsof contaminating. Using each of the thus obtained total RNA samples,single-stranded cDNAs are synthesized by SUPERSCRIPT™ PreamplificationSystem for First Strand cDNA System (manufactured by Life Technologies)by using oligo(dT) primers or random primers. Examples ofsingle-stranded cDNAs include single-stranded cDNAs prepared from ahuman neuroblastoma cell line SK-N-MC by the above method.

The cDNA library can be prepared by the usual method. Examples of thecDNA library preparation method include the method described inMolecular Cloning, 2nd Ed., Current Protocols in Molecular Biology, DNACloning 1: Core Techniques, A Practical Approach, 2nd Ed., OxfordUniversity Press (1995) or the like, and the method in which acommercially available kit such as SuperScript Plasmid System for cDNASynthesis and Plasmid Cloning (manufactured by GIBCO BRL) or ZAP-cDNASynthesis Kit (manufactured by STRATAGENE) is used, and the like. A cDNAlibrary derived from various organs or various cells can also beobtained by purchasing a commercially available product.

As the cloning vector for preparing a cDNA library, any one of phagevectors, plasmid vectors and the like can be used, so long as it canreplicate autonomously in Escherichia coli K12. Examples include ZAPExpress [manufactured by STRATAGENE, Strategies, 5, 58 (1992)], pBlue IISK(+) [Nucleic Acids Research, 17, 9494 (1989)], λZAP II (manufacturedby STRATAGENE), λgt10 [DNA Cloning, A Practical Approach, 1, 49(1985)],λTriplEx (manufactured by Clontech), λExCell (manufactured byPharmacia), pT7T318U (manufactured by Pharmacia), pcD2 [Mol. Cell.Biol., 3, 280 (1983)], pUC18 [Gene, 33, 103 (1985)], pAMo [J. Biol.Chem., 268, 22782 (1993), alias pAMoPRC3Sc (Japanese Published.Unexamined Patent Application No. 336963/93)] and the like.

As the host microorganism, any microorganism can be used, so long as itbelongs to Escherichia coli. Specifically, Escherichia coli XL1-BlueMRF′ [manufactured by STRATAGENE, Strategies, 5, 81 (1992)], Escherichiacoli C600 [Genetics, 39, 440 (1954)], Escherichia coli Y1088 [Science,222, 778 (1983)], Escherichia coli Y1090 [Science, 222, 778 (1983)],Escherichia coli NM522 [J. Mol. Biol., 166, 1 (1983)], Escherichia coliK802 [J. Mol. Biol., 16, 118 (1966)], Escherichia coli JM105 [Gene, 38,275 (1985)], Escherichia coli SOLR™ Strain (available from STRATAGENE),Escherichia coli LE392 (Molecular Cloning, 2nd Ed) and the like.

As the cDNA library, mention may be made of a cDNA library prepared asfollows.

A cDNA library is prepared by synthesizing cDNAs from human gastricmucosa poly(A)+ RNA by using cDNA Synthesis System (manufactured byGIBCO BRL), adding an EcoRI-NotI-SalI adapter (Super Choice System forcDNA Synthesis; manufactured by GIBCO BRL) to their both termini,inserting them into the EcoRI site of a cloning vector λZAP II (λZAPII/EcoRI/CIAP Cloning Kit, manufactured by STRATAGENE), and thencarrying out in vitro packaging by using Gigapack III Gold PackagingExtract manufactured by STRATAGENE. Alternatively, a commerciallyavailable cDNA library can be used.

Based on the nucleotide sequence of a candidate gene found by the database search, primers specific for the gene are designed and PCR iscarried out by using the thus obtained single-stranded cDNAs or a cDNAlibrary as the templates. When an amplified fragment is obtained, thefragment is subcloned into an appropriate plasmid. The subcloning can becarried out by inserting the amplified DNA fragment directly, or afterits treatment with a restriction enzyme or DNA polymerase, into a vectorin the usual way. Examples of the vector include pBlue SK(−), pBlue IISK(+) (both manufactured by STRATAGENE), pDIRECT [Nucleic AcidsResearch, 18, 6069 (1990)], pCR-Amp SK(+) [manufactured by Stratagene,Strategies, 5, 6264 (1992)], pT7Blue (manufactured by Novagen), pCR II[manufactured by Invitrogen; Biotechnology, 9, 657 (1991)], pCR-TRAP(manufactured by Genehunter), pNoTA_(T7) (manufactured by 5′→3′) and thelike.

Acquisition of the DNA fragment of interest is confirmed by determiningthe nucleotide sequence of the subcloned PCR amplification fragment. Thenucleotide sequence can be determined by the generally used nucleotidesequence analyzing method such as the dideoxy method of Sanger et al.[Proc. Natl. Acad. Sci. USA, 74, 5463 (1997)] or by using the nucleotidesequence analyzing apparatus such as 373A DNA sequencer (manufactured byPERKIN ELMER) or the like.

By carrying out colony hybridization or plaque hybridization (MolecularCloning, 2nd Ed) for the cDNA library prepared in the above by using theDNA fragment as a probe, cDNA having a possibility of encoding a proteinhaving homology with β3Gal-T1 at the amino acid level can be obtained.As the probe, the DNA fragment labeled with an isotope or digoxigenincan be used.

The nucleotide sequence of the DNA obtained by the above method can bedetermined by inserting the DNA fragment as such or after its digestionwith an appropriate restriction enzyme or the like into a vector by thegeneral method described in Molecular Cloning, 2nd Ed or the like, andthen analyzing it by the generally used nucleotide sequence analyzingmethod such as the dideoxy method of Sanger et al. [Proc. Natl. Acad.Sci. USA, 74, 5463 (1997)] or using a nucleotide sequence analyzingapparatus such as 373A DNA sequencer (manufactured by PERKIN ELMER) orthe like.

Examples of the DNA obtained by the method include DNAs encoding thepolypeptide represented by SEQ ID NO:1, 2, 3 or 4. Specific examplesinclude DNAs having the nucleotide sequence represented by SEQ ID NO:5,6, 7 or 8. Examples of a plasmid containing the DNA of SEQ ID NO:5include pAMo-G3 and pBS-G3. Examples of a plasmid containing the DNA ofSEQ ID NO:6 include pAMo-G4 and pBS-G4. Examples of a plasmid containingthe DNA of SEQ ID NO:7 include pAMo-G4-2 and pBS-G4-2. Examples of aplasmid containing the DNA of SEQ ID NO:8 include pAMo-G7 and pT7B-G7.

The DNA of interest encoding a polypeptide comprised of an amino acidsequence in which one or more amino acids are deleted, substituted oradded when compared with the amino acid sequence represented by SEQ IDNO:1, 2, 3 or 4 can be obtained by selecting a DNA which hybridizes withthe DNA obtained by the above method under stringent conditions. Forexample, the DNA of interest can be obtained by screening for a cDNAlibrary derived from other species (mouse, rat, calf, monkey or thelike).

A DNA which is hybridizable under stringent conditions is a DNA obtainedby carrying out colony hybridization, plaque hybridization, Southernhybridization or the like using the DNA obtained in the above as aprobe. Examples include a DNA which can be identified by carrying outhybridization at 65° C. in the presence of 0.7 to 1.0 M sodium chlorideusing a filter to which colony- or plaque-derived DNA samples areimmobilized and then washing the filter at 65° C. with 0.1 to 2-foldconcentrated SSC solution (1-fold concentrated SSC solution contains 150mmol/l sodium chloride and 15 mmol/l sodium citrate). The hybridizationcan be carried out in accordance with the method described in MolecularCloning, 2nd Ed., Current Protocols in Molecular Biology, John Wiley &Sons (1987-1997) (hereinafter referred to as “Current Protocols inMolecular Biology”), DNA Cloning 1: Core Techniques, A PracticalApproach, 2nd Ed.; Oxford University Press (1995) or the like. Examplesof the hybridizable DNA include a DNA having at least 60% or more ofhomology, preferably a DNA having 80% or more of homology, morepreferably a DNA having 95% or more of homology, with the DNA obtainedin the above, when calculated using BLAST [J. Mol. Biol., 215, 403(1990)], FASTA [Methods in Enzymology, 183, 63-98 (1990)] or the like.

The DNA of interest encoding a polypeptide comprised of an amino acidsequence in which one or more amino acids are deleted, substituted oradded can be obtained by using a site-directed mutagenesis methoddescribed in Molecular Cloning, 2nd Ed., Current Protocols in MolecularBiology, Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci.USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13,4431 (1985), Proc. Natl. Acad. Sci. USA, 82, 488 (1985) or the like, forexample by introducing the site-directed mutagenesis into a DNA encodinga polypeptide having the amino acid sequence represented by SEQ ID NO:1.The number of amino acids to be deleted, substituted or added is notparticularly limited, but limited within such a range that polypeptidesof the present invention do not include known polypeptides, and from 1to several tens, particularly from 1 to several, of amino acids arepreferable. Also, in order to maintain aβ1,3-N-acetylglucosaminyltransferase activity of the polypeptide of thepresent invention, it is preferable that it has 60% or more, generally80% or more, particularly 95% or more, of homology with, for example,the amino acid sequence represented by SEQ ID NO:1.

The DNA of interest can also be prepared by chemically synthesizing aDNA encoding the polypeptide based on the determined amino acid sequenceof the novel glycosyltransferase polypeptide. Chemical synthesis of theDNA can be carried out by using a DNA synthesizer manufactured byShimadzu which employs the thiophosphite method, a DNA synthesizer model392 manufactured by PERKIN ELMER which employs the phosphoamiditemethod, and the like.

The DNA of interest can also be prepared by carrying out PCR usingoligonucleotides described below as a sense primer and an antisenseprimer, and by using cDNAs prepared from a cell expressing mRNAcomplementary to these DNA molecules as the template.

Oligonucleotides, such as antisense oligonucleotide, senseoligonucleotide and the like, having a partial sequence of the DNA ofthe present invention can be prepared by using the DNA and DNA fragmentsof the present invention obtained by the above method, in accordancewith the general method described in Molecular Cloning, 2nd Ed or thelike, or by using a DNA synthesizer based on the nucleotide sequenceinformation on the DNA.

Examples of the oligonucleotide include a DNA having a sequenceidentical to continuous 5 to 60 nucleotides in a nucleotide sequencecontained in the above DNA. Specific examples include a DNA having asequence identical to continued 5 to 60 nucleotides in the nucleotidesequence represented by SEQ ID NO:5, 6, 7 or 8 or a DNA having asequence complementary to the above DNA. When used as a sense primer andan antisense primer, the above oligonucleotides in which the meltingtemperature (Tm) and the number of bases are not significantly differentfrom each other are preferable. Specifically, oligonucleotides havingthe nucleotide sequences shown in SEQ ID NOS:9-20 can be exemplified.

In addition, derivatives of these oligonucleotides (hereinafter referredto as “oligonucleotide derivatives”) can also be used as theoligonucleotides of the present invention.

Examples of the oligonucleotide derivatives include oligonucleotidederivatives in which a phosphodiester bond in the oligonucleotide isconverted into a phosphorothioate bond, oligonucleotides derivative inwhich a phosphodiester bond in the oligonucleotide is converted into anN3′-P5′ phosphoamidate bond, oligonucleotide derivatives in which riboseand a phosphodiester bond in the oligonucleotide are converted into apeptide-nucleic acid bond, oligonucleotide derivatives in which uracilin the oligonucleotide is substituted with C-5 propynyluracil,oligonucleotide derivatives in which uracil in the oligonucleotide issubstituted with C-5 thiazoleuracil, oligonucleotide derivatives inwhich cytosine in the oligonucleotide is substituted with C-5propynylcytosine, oligonucleotide derivatives in which cytosine in theoligonucleotide is substituted with phenoxazine-modified cytosine,oligonucleotide derivatives in which ribose in the oligonucleotide issubstituted with 2′-O-propylribose, oligonucleotide derivatives in whichribose in the oligonucleotide is substituted with2′-methoxyethoxyribose, and the like [Cell Technology, 16, 1463 (1997)].

(2) Determination of an Activity of a Polypeptide Encoded by theObtained DNA

An expression plasmid is constructed by inserting the DNA obtained inthe above manner into an expression vector. After introducing theplasmid into an appropriate animal cell, whether or not the DNA isinvolved in the synthesis of sugar chains can be examined according tothe fluorescence activated cell sorter (hereinafter referred to as“FACS”) analysis by using an antibody or lectin which specifically bindsto a sugar chain (poly-N-acetyllactosamine sugar chain, sialyl Lewis asugar chain or sialyl Lewis c sugar chain).

Any expression vector can be used, so long as the cDNA can be insertedand the vector can function in an animal cell. Examples includepcDNAI/Amp, pcDNAI, pCDM8 (both available from Funakoshi), pAGE107[(Japanese Published Unexamined Patent Application No. 22979/92,Cytotechnology, 3, 133 (1990)], pREP4 (manufactured by Invitrogen),pAGE103 [J. Biochem., 101, 1307 (1987)], pAMo, pAMoA [[J. Biol. Chem.,268, 22782 (1993), alias pAMoPRSA (Japanese Published Unexamined PatentApplication No. 336963/93)], pAS3-3 (Japanese Published UnexaminedPatent Application No. 227075/90) and the like.

A transformed cell is obtained by introducing the cDNA-insertedexpression vector into an animal cell which can be used in selecting thecDNA of interest.

Regarding the method for introducing this expression vector, any methodfor introducing DNA into animal cells can be used. Examples includeelectroporation method [Cytotechnology, 3, 133(1990)], calcium phosphatemethod (Japanese Published Unexamined Patent Application No. 227075/90),lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)] and thelike.

Examples of the animal cell include Namalwa cell as a human cell,Namalwa KJM-1 cell as a sub-line of Namalwa cell, 293 cell, COS cell asa monkey cell, CHO cell as a Chinese hamster cell and HBT5637 (JapanesePublished. Unexamined Patent Application No. 299/88). Among these,Namalwa cell and Namalwa KJM-1 cell are preferred.

The thus obtained transformed cell is cultured by the general method.

Specifically, the following culturing method for transformants can beexemplified.

Examples of the medium for culturing the cell include RPMI 1640 medium[The Journal of the American Medical Association, 199, 519 (1967)],Eagle's MEM medium [Science, 122, 501 (1952)], DMEM medium [Virology, 8,396 (1959)], 199 medium [Proceeding of the Society for the BiologicalMedicine, 73, 1 (1950)] and a medium prepared by adding fetal bovineserum or the like to any of these media, and the like.

Culturing is carried out generally at pH 6 to 8 and at 30 to 40° C. inthe presence of 5% CO₂ for 1 to 7 days. Also, if necessary, antibioticssuch as kanamycin, penicillin and the like may be added to the medium.

The transformed cell obtained by the culturing is subjected tofluorescence staining using an antibody or lectin which specificallybinds to a sugar chain (poly-N-acetyllactosamine sugar chain, sialylLewis a sugar chain or sialyl Lewis c sugar chain) and then analyzed byusing FACS. When a poly-N-acetyllactosamine sugar chain is produced, inan increased amount in comparison with a transformed cell into which acontrol plasmid is introduced, it can be considered that the novelpolypeptide encoded by the DNA has aβ1,3-N-acetylglucosaminyltransferase activity which involved in thesynthesis of the poly-N-acetyllactosamine sugar chain. On the otherhand, when a sialyl Lewis a sugar chain or sialyl Lewis c sugar chain isproduced in an increased amount, it can be considered that the novelpolypeptide encoded by the DNA has a β-1,3-galactosyltransferaseactivity involved in the synthesis of the sialyl Lewis a sugar chain orsialyl Lewis c sugar chain.

As a antibody or lectin which recognizes a poly-N-acetyllactosaminesugar chain, any substance capable of recognizing spoly-N-acetyllactosamine sugar chain can be used. Examples of theantibody which recognizes a poly-N-acetyllactosamine sugar chain includeanti-i antibody. Examples of the lectin which recognizespoly-N-acetyllactosamine sugar chain include pokeweed mitogen (referredto as “PWM”), Lycopersicon esculentum (tomato) agglutinin (referred toas “LEA”) and Datura stramonium agglutinin (referred to as “DSA”) [J.Biol. Chem., 282, 8179 (1987), J. Biol. Chem., 259, 6253 (1984), J.Biol. Chem., 262, 1602 (1987), Carbohydr. Res., 120, 187 (1983),Carbohydr. Res., 120, 283-292 (1983), Glycoconjugate J., 7, 323 (1990)].

As an anti-sialyl Lewis a sugar chain antibody or anti-sialyl Lewis csugar chain antibody, any antibody which reacts with a sialyl Lewis asugar chain or sialyl Lewis sugar chain can be used. Examples include19-9 (manufactured by Fujirebio) and KM231 (manufactured by Kyowa Medex)as the anti-sialyl Lewis a sugar chain antibodies, and DU-PAN-2(manufactured by Kyowa Medex) as the anti-sialyl Lewis c sugar chainantibody.

Also, a β1,3-N-acetylglucosaminyltransferase activity can be measuredusing a cell extract of the above transformed cell in accordance withthe known measuring methods [J. Biol. Chem., 268, 27118 (1993), J. Biol.Chem., 267, 2994 (1992), J. Biol. Chem., 263, 12461 (1988), Jpn. J. Med.Sci, Biol., 42, 77 (1989)]. When the amount ofβ1,3-N-acetylglucosaminyltransferase activity is increased in comparisonwith a transformed cell into which a control plasmid is introduced, itcan be considered that the novel polypeptide encoded by the DNA has aβ1,3-N-acetylglucosaminyltransferase activity.

Also, a β1,3-galactosyltransferase activity of the polypeptide of thepresent invention can be measured in accordance with the known measuringmethods [J. Biol. Chem., 2-58, 9893-9898 (1983), J. Biol. Chem., 262,15649 (1987), Archi. Biochem. Biophys., 270, 630 (1989), Archi. Biochem.Biophys., 274, 14 (1989), Japanese Published Unexamined PatentApplication No. 181759/94, J. Biol. Chem., 273, 58 (1998), J. Biol.Chem., 273, 433 (1998), J. Biol. Chem., 273, 12770 (1998), J. Biol.Chem., 274, 12499 (1999)].

In the above manner, the activity of the obtained novel polypeptideencoded by the novel cDNA can be found.

(3) Production of a Novel β1,3-N-acetylglucosaminyltransferasePlypeptide

In order to produce the polypeptide of the present invention byexpressing the DNA of the present invention obtained by the methoddescribed in the above in a host cell, the methods described inMolecular Cloning, 2nd Ed., Current Protocols in Molecular Biology,Supplements 1 to 38 (Current Protocols in Molecular Biology) and thelike can be used.

That is, the polypeptide of the present invention can be produced byconstructing a recombinant vector in which the DNA of the presentinvention is inserted into downstream of a promoter in an appropriateexpression vector, introducing the vector into a host cell to therebyobtain a transformant capable of expressing the polypeptide of thepresent invention, and then culturing the transformant.

As the host cell, any one of prokaryotic cells, yeast cells, animalcells, insect cells, plant cells and the like can be used, so long asthe gene of interest is expressed in the cell.

The expression vector to be used is a vector which can autonomouslyreplicate in the above host cells or can be integrated into chromosomeand contains a promoter at a position suitable for the transcription ofthe novel β1,3-N-acetylglucosaminyltransferase gene.

When a prokaryotic organism such as a bacterium or the like is used asthe host cell, it is preferable that the expression vector of the novelβ1,3-N-acetylglucosaminyltransferase gene can autonomously replicate inthe prokaryotic organism and is constituted from a promoter, a ribosomebinding sequence, the novel β1,3-N-acetylglucosaminyltransferase geneand a transcription termination sequence. A gene which controls thepromoter may also be contained.

Examples of the expression vector include pBTrp2, pBTac1, pBTac2 (allavailable from Boehringer-Mannheim), pSE280 (manufactured byInvitrogen), pGEMEX-1 (manufactured by Promega), pQE-8 (manufactured byQIAGEN), pKYP10 (Japanese Published Unexamined Patent Application No.110600/83), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSA1 [Agric.Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82,4306 (1985)], pBlue II SK(−) (manufactured by STRATAGENE), pTrs30 (FERMBP-5407), pTrs32 (FERM BP-5408), pGHA2 (FERM BP-400), pGKA2 (FERMB-6798), pTerm2 (Japanese Published Unexamined Patent Application No.22979/91, U.S. Pat. No. 4,686,191, U.S. Pat. No. 4,939,094, U.S. Pat.No. 5,160,735), pKK233-2 (manufactured by Pharmacia), pGEX (manufacturedby Pharmacia), pET system (manufactured by Novagen), pSupex, pUB110,pTP5, pC194, pTrxFus (manufactured by Invitrogen), pMAL-c2 (manufacturedby New England Biolabs) and the like.

As the promoter, any promoter may be used so long as it can be expressedin host cells such as Escherichia coli and the like. Examples includepromoters, such as trp promoter (Ptrp), lac promoter (Plac), P_(L)promoter, P_(R) promoter and the like, derived from Escherichia coli,phage and the like, and SPO1 promoter, SPO2 promoter, penP promoter andthe like. In addition, artificially designed and modified promoters suchas a promoter in which two Ptrp are linked in tandem (Ptrp×2), tacpromoter, lacT7 promoter, letI promoter and the like can also be used.

As the ribosome binding sequence, it is preferable to use a plasmid inwhich the space between the Shine-Dalgarno sequence and initiation codonis ajusted at an appropriate distance (e.g., 6 to 18 nucleotides).

Although a transcription termination sequence is not always necessaryfor the expression of the DNA of the present invention, it is preferableto arrange the transcription termination sequence just below thestructural gene.

Examples of the host cell include microorganisms belonging to the genusEscherichia, the genus Serratia, the genus Bacillus, the genusBrevibacterium, the genus Corynebacterium, the genus Microbacterium, thegenus Pseudomonas and the like, such as Escherichia coli XL1-Blue,Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coliMC1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichiacoli JM109, Escherichia coli HB101, Escherichia coli No. 49, Escherichiacoli W3110, Escherichia coli NY49, Escherichia coli BL21(DE3),Escherichia coli BL21(DE3)pLysS, Escherichia coli HMS174(DE3),Escherichia coli HMS174(DE3)pLysS, Serratia ficaria, Serratia fonticola,Serratia liquefaciens, Serratia marcescens, Bacillus subtilis, Bacillusamyloliquefaciens, Brevibacterium ammoniagenes, Brevibacteriumimmariophilum ATCC 14068, Brevibacterium saccharolyticum ATCC 14066,Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum ATCC14067, Corynebacterium glutamicum ATCC 13869, Corynebacteriumacetoacidophilum ATCC 13870, Microbacterium ammoniaphilum ATCC 15354,Pseudomonas sp. D-0110 and the like.

Regarding the method for introducing the recombinant vector, any methodin which DNA can be introduced into the above host cells can be used.Examples include electroporation method [Nucleic Acids Res., 16, 6127(1988)], a method which uses calcium ion [Proc. Natl. Acad. Sci. USA,69, 2110 (1972)], protoplast method (Japanese Published UnexaminedPatent Application No. 248394/88), the methods described in Gene, 17,107 (1982) and Molecular & General Genetics, 168, 111 (1979) and thelike.

When yeast is used as the host cell, YEp13 (ATCC 37115), YEp24 (ATCC37051), YCp50 (ATCC 37419), pHS19, pHS15 and the like can, for example,be used as the expression vector.

Any promoter can be used so long as it can be expressed in yeast.Examples include PH05 promoter, PGK promoter, GAP promoter, ADHpromoter, gal 1 promoter, gal 10 promoter, heat shock protein promoter,MF α1 promoter, CUP 1 promoter and the like.

Examples of the host cell include yeast belonging to the genusSaccharomyces, the genus Schizosaccharomyces, the genus Kluyveromyces,the genus Trichosporon, the genus Schwanniomyces and the like. Specificexamples include Saccharomyces cerevisiae, Schizosaccharomyces pombe,Kluyveromyces lactis, Trichosporon pullulans, Schwanniomyces alluviusand the like.

Regarding the method for introducing the recombinant vector, any one ofthe methods for introducing DNA into yeast can be used. Examples includeelectroporation method [Methods in Enzymology, 194, 182 (1990)],spheroplast method [Proc. Natl. Acad. Sci. USA, 84, 1929 (1978)],lithium acetate method [J. Bacteriol., 153, 163 (1983)], a methoddescribed in Proc. Natl. Acad. Sci. USA, 75, 1929 (1978) and the like.

When an animal cell is used as the host, pcDNAI/Amp, pcDNAI, pCDM8,pAGE107, pREP4, pAGE103, pAMo, pAMoA, pAS3-3 and the like can beexemplified as the expression vector.

Any promoter allowing the expression in animal cells can be used.Examples include the promoter of IE (immediate early) gene ofcytomegalovirus (CMV), the early promoter of SV40, the long terminalrepeat promoter of Moloney murine leukemia virus, the promoter ofretrovirus, the heat shock promoter, the SRα promoter, the promoter ofmetallothione in and the like. Also, the enhancer of the IE gene ofhuman CMV may be used together with the promoter.

Examples of the host cell include a mouse myeloma cell, a rat myelomacell, a mouse hybridoma cell, CHO cell as Chinese hamster cell, BHKcell, African green monkey kidney cell, Namalwa cell or Namalwa KJM-1cell as a human cell, a human fetal kidney cell, a human leukemia cell,HBT5637 (Japanese Published Unexamined Patent Application No. 299/88), ahuman colon cancer cell line and the like.

The mouse myeloma cells include SP2/0, NS0 and the like. The rat myelomacells include YB2/0 and the like. The human fetal kidney cells includeHEK293, 293 and the like. The human leukemia cells include BALL-1 andthe like. The African green monkey kidney cells include COS-1, COS-7 andthe like. The human large bowel cancer cell lines include HCT-15 and thelike.

Regarding the method for introducing the recombinant vector, any one ofthe methods for introducing, DNA into animal cells can be used. Examplesinclude electroporation method [Cytotechnology, 3, 133 (1990)], calciumphosphate method (Japanese Published Unexamined Patent Application No.227075/90), lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413(1987)], the method described in Virology, 52, 456 (1973) and the like.Preparation of a transformant and its culturing can be carried out inaccordance with the method described in Japanese Published UnexaminedPatent Application No. 227075/90 or Japanese Published Unexamined PatentApplication No. 257891/90.

When insect cells are used as the host, the polypeptide can be expressedby the method described, for example, in Baculovirus Expression Vectors,A Laboratory Manual, W.H. Freeman and Company, New York (1992),Molecular Biology, A Laboratory Manual, Current Protocols in MolecularBiology, Supplements 1 to 38 or Bio/Technology, 6, 47 (1988).

That is, the polypeptide can be expressed by simultaneouslycotransfecting a recombinant gene transfer vector and a baculovirus intoan insect cell to obtain a recombinant virus in an insect cell culturesupernatant and then infecting the insect cell with the recombinantvirus.

Examples of the gene transfer vector to be used in this method includepVL1392, pVL1393, pBlueBacIII (all manufactured by Invitrogen).

Examples of the baculovirus include Autographa californica nuclearpolyhedrosis virus with which insects of the family Barathra areinfected and the like.

Examples of the insect cells include Spodoptera frugiperda oocyte,Trichoplusia ni oocyte, Bombyx mori oocyte-derived culturing cell andthe like.

The Spodoptera frugiperda oocytes include Sf9 and Sf21 (BaculovirusExpression Vectors, A Laboratory Manual) and the like. The Trichoplusiani oocytes include High 5, BTI-TN-5B1-4 (manufactured by Invitrogen) andthe like. The Bombyx mori oocyte culture cells include Bombyx mori N4and the like.

Examples of the method for the simultaneous cotransfection of the aboverecombinant gene transfer vector and the above baculovirus for thepreparation of the recombinant virus include calcium phosphate method(Japanese Published Unexamined Patent Application No. 227075/90),lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)] and thelike.

Also, DNA can be introduced into insect cells by the same method as thatas used for introducing DNA into animal cells. Examples includeelectroporation method [Cytotechnology, 3, 133 (1990)], calciumphosphate method (Japanese Published Unexamined Patent Application No.227075/90), lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413(1987)] and the like.

When a plant cell or a plant is used as the host, the polypeptide can beproduced in accordance with known methods [Tissue Culture, 20 (1994),Tissue Culture 21 (1995), Trends in Biotechnology, 15, 45 (1997)].

As the promoter to be used in the gene expression, any promoter can beused, so long as it can function in plant cells. Examples includecauliflower mosaic virus (CaMV) 35S promoter, rice actin 1 promoter andthe like. Also, the gene expression efficiency can be improved byinserting intron 1 of corn alcohol dehydrogenase gene or the likebetween the promoter and the gene to be expressed.

Examples of the host cell include plant cells such as potato, tobacco,corn, rice, rape, soybean, tomato, wheat, barley, rye, alfalfa, flax andthe like. As the method for introducing a recombinant vector, any methodfor introducing DNA into plant cells can be used. Examples include amethod using Agrobacterium (Japanese Published Unexamined PatentApplication No. 140885/84, Japanese Published Unexamined PatentApplication No. 70080/85, WO 94/00977), electroporation method[Cytotechnology, 3, 133 (1990), Japanese Published Unexamined PatentApplication No. 251887/85], a method using a particle gun (gene gun)(Japanese Patent No. 2606856, Japanese Patent No. 2517813) and the like.

A cell or organ of the gene-introduced plant can be cultured in a largeamount by using a jar fermentor. Also, a gene-introduced plant(transgenic plant) can be constructed by re-differentiating thegene-introduced plant cell.

The polypeptide of the present invention can also be produced by usingan animal. For example, the polypeptide of the present invention can beproduced in a gene-introduced animal in accordance with known methods[American Journal of Clinical Nutrition, 63, 639S (1996), AmericanJournal of Clinical Nutrition, 63, 627S (1996), Bio/Technology, 9, 830(1991)].

Any promoter which can be expressed in an animal can be used, forexample, mammary gland cell-specific promoters such as α-caseinpromoter, β-lactoglobulin promoter, whey acidic protein promoter and thelike are suitably used.

The polypeptide of the present invention can be produced by culturing atransformant derived from a microorganism, animal cell or plant cellhaving a recombinant vector into which a DNA encoding the polypeptide isinserted, in accordance with a general culturing method, to therebyproduce and accumulate the polypeptide, and then recovering thepolypeptide from the resulting culture mixture.

When the transformant is an animal or plant, the polypeptide can beproduced by rearing or cultivating it in accordance with a generalrearing or cultivating method to thereby produce and accumulate thepolypeptide, and then recovering the polypeptide from the animal orplant.

That is, in an animal, the polypeptide having the novelβ1,3-N-acetylglucosaminyltransferase activity can be obtained by, forexample, rearing a non-human transgenic animal retaining the DNA of thepresent invention to thereby produce and accumulate the polypeptidehaving the novel β1,3-N-acetylglucosaminyltransferase activity encodedby the recombinant DNA, in the animal, and then recovering thepolypeptide from the animal. Examples of the production and accumulationregion in the animal include milk, eggs and the like.

In a plant, the polypeptide having the novelβ1,3-N-acetylglucosaminyltransferase activity can be obtained by, forexample, cultivating a transgenic plant having the DNA of the presentinvention to thereby produce and accumulate the polypeptide having thenovel β1,3-N-acetylglucosaminyltransferase activity encoded by therecombinant DNA, in the plant, and then recovering the polypeptide fromthe plant.

When the transformant for use in the production of the polypeptide ofthe present invention is prokaryote such as Escherichia coli or thelike, or eukaryote such as yeast or the like, the medium for culturingsuch an organism may be either a natural medium or a synthetic medium,so long as it contains carbon sources, nitrogen sources, inorganic saltsand the like which can be assimilated by the organism and can be usedfor the efficient culture of the transformant.

The carbon sources include those which can be assimilated by thetransformant. Examples include carbohydrates such as glucose, fructose,sucrose, molasses containing them, starch, starch hydrolysate and thelike; organic acids such as acetic acid, propionic acid and the like;alcohols such as ethanol, propanol and the like.

Examples of the nitrogen sources include ammonia, various ammonium saltsof inorganic acids and organic acids such as ammonium chloride, ammoniumsulfate, ammonium acetate, ammonium phosphate and the like; othernitrogen-containing compounds, as well as peptone, meat extract, yeastextract, corn steep liquor, casein hydrolysate, soybean meal and soybeanmeal hydrolysate and various fermented cells and hydrolysates thereof.

Examples of the inorganic sals include potassium dihydrogen phosphate,dipotassium hydrogen phosphate, magnesium phosphate, magnesium sulfate,sodium chloride, ferrous sulfate; manganese sulfate, copper sulfate,calcium carbonate and the like.

Culturing is carried out under aerobic condition such as shakingculture, submerged spinner culture under aeration or the like. Theculturing temperature is preferably from 15 to 40° C., and the culturingtime is generally from 16 to 96 hours. During culturing, the pH iscontrolled at 3.0 to 9.0. The pH is adjusted using an inorganic ororganic acid, an alkali solution, urea, calcium carbonate, ammonia andthe like.

If necessary, antibiotics such as ampicillin, tetracycline and the likemay be added to the medium during culturing.

When a microorganism transformed with an expression vector obtainedusing an inducible promoter as the promoter is cultured, an inducer maybe added to the medium, if necessary. For example,isopropyl-β-D-thiogalactopyranoside (IPTG) or the like may be added tothe medium when a microorganism transformed with an expression vectorobtained using lac promoter is cultured, or indoleacrylic acid (IAA) orthe like may be added to the medium when a microorganism transformedwith an expression vector obtained by using trp promoter is cultured.

When the transformant for use in the production of the polypeptide ofthe present invention is an animal cell, generally-used media such asRPMI 1640 medium [The Journal of the American Medical Association, 199,519 (1967)], Eagle's MEM medium [Science, 122, 501 (1952)], DMEM medium[Virology, 8, 396 (1959)], 199 Medium [Proceeding of the Society for theBiological Medicine, 73, 1 (1950)] or any one of these media furthersupplemented with fetal calf serum or the like can be used.

Culturing is carried out generally at pH 6 to 8 and at 30 to 40° C. inthe presence of 5% CO₂ for 1 to 7 days. If necessary, antibiotics suchas kanamycin, penicillin and the like may be added to the medium duringculturing.

Regarding the medium for use in culturing of a transformant obtainedusing an insect cell as the host, usually used TNM-FH medium(manufactured by PharMingen), Sf-900 II SFM medium (manufactured byGIBCO BRL), ExCell 400 or ExCell 405 (both manufactured by JRHBiosciences), Grace's Insect Medium [Nature, 195, 788 (1962)] or thelike can be used.

Culturing is carried out at pH 6 to 7 and at 25 to 30° C. for 1 to 5days. In addition, antibiotics such as gentamicin and the like may beadded to the medium during culturing, if necessary.

Regarding the method for expression of the gene, in addition to the caseof expressing the full-length polypeptide, it can also be expressed as apartial polypeptide containing a region having theβ1,3-N-acetylglucosaminyltransferase activity. In general, aglycosyltransferase has the topology of type II membrane protein andcomprises an N-terminal cytoplasmic region containing several to severalten amino acids, a membrane-binding region having a highly hydrophobicamino acid sequence, a stem region containing several to several tenamino acids and the remaining C-terminal part comprising most of thepolypeptide and containing a catalytic region. It is considered that thestem region and the remaining C-terminal part comprising most of thepolypeptide and containing a catalytic region are exposed to the Golgibody cavity. The boundary between the stem region and catalytic regioncan be experimentally examined by preparing an N-terminus-deletedpolypeptide and examining the degree of the deletion by which theactivity disappears. On the other hand, the amino acid sequence of thestem region and catalytic region can be presumed by comparing with thatof a similar glycosyltransferase having information on the stem regionand catalytic region.

As for the novel β1,3-N-acetylglucosaminyltransferase of the presentinvention, it was presumed that the polypeptide having the amino acidsequence represented by SEQ ID NO:1 comprises an N-terminal cytoplasmicregion containing 9 amino acids, a subsequent membrane-binding regionrich in hydrophobic nature containing 19 amino acids, a stem regioncontaining at least 12 amino acids and the remaining C-terminal partcomprising most of the polypeptide and containing a catalytic region,each of the polypeptides having the amino acid sequences represented bySEQ ID NOS:2 and 3 comprises an N-terminal cytoplasmic region containing11 amino acids, a subsequent membrane-binding region rich in hydrophobicnature containing 21 amino acids, a stem region containing at least 12amino acids and the remaining C-terminal part comprising most of thepolypeptide and containing a catalytic region, and the polypeptidehaving the amino acid sequence represented by SEQ ID NO:4 comprises anN-terminal cytoplasmic tail region containing 29 amino acids, asubsequent membrane-binding region rich in hydrophobic nature containing20 amino acids, a stem region containing at least 12 amino acids and theremaining C-terminal part comprising most of the polypeptide andcontaining a catalytic region.

The stem region was presumed based on the comparison of the homology ofthe amino acid sequence with those of otherβ1,3-N-acetylglucosaminyltransferases and a β1,3-galactosyltransferase,and the information on the stem regions of otherβ1,3-N-acetylglucosaminyltransferases and a β1,3-galactosyltransferase(Example 4 in this specification, Japanese Published Unexamined PatentApplication No. 181759/94). Accordingly, it is considered that apolypeptide containing an amino acid sequence of positions 41-397 of SEQID NO:1, polypeptides containing an amino acid sequence of positions45-372 of SEQ ID NOS:2 and 3 and a polypeptide containing an amino acidsequence of positions 62-378 of SEQ ID NO:4 contain catalytic regions.

In addition to its direct expression, the above full-length polypeptideor partial polypeptide containing a region having aβ1,3-N-acetylglucosaminyltransferase activity (catalytic region) canalso be expressed as a secreted protein or a fusion protein inaccordance with the method described in Molecular Cloning, 2nd Ed. orthe like. Examples of the protein to be fused include β-galactosidase,protein A, IgG binding region of protein A, chloramphenicolacetyltransferase, poly(Arg), poly(Glu), protein G, maltose-bindingprotein, glutathione S-transferase, polyhistidine chain (His-tag), Speptide, DNA binding protein domain, Tac antigen, thioredoxin, greenfluorescent protein, FLAG peptide, epitopes of antibodies of interestand the like [Jikken Igaku, 13, 469 (1995)].

When the polypeptide of the present invention is produced in a host cellor on the outer membrane of a host cell, the polypeptide can bepositively secreted extracellulary in accordance with the method ofPoulson et al. [J. Biol. Chem., 264, 17619 (1989)], the method of Loweet al. [Proc. Natl. Acad. Sci. USA, 86, 8227 (1989), Genes Develop., 4,1288 (1990)], or the methods described in Japanese Published UnexaminedPatent Application No. 336963/93, WO 94/23021 and the like.

That is, the polypeptide of the present invention can be positivelysecreted by expressing it in a form in which a signal peptide is addedto the upstream of a polypeptide containing an active region of thepolypeptide of the present invention, in accordance with recombinant DNAtechniques.

Specifically, it is considered that the polypeptide of the presentinvention can be positively secreted extracellulary by adding a signalpeptide to the upstream of a polypeptide having an amino acid sequencepresumably containing a catalytic region and expressing the product. Inaddition, a tag for use in the purification and detection can be addedbetween the signal peptide and the catalytic region, or to theC-terminus of a polypeptide containing the catalytic region.

Examples of the tag for use in the purification and detection includeβ-galactosidase, protein A, IgG binding region of protein A,chloramphenicol acetyltransferase, poly(Arg), poly(Glu), protein G,maltose-binding protein, glutathione S-transferase, polyhistidine chain(His-tag), S peptide, DNA binding protein domain, Tac antigen,thioredoxin, green fluorescent protein, FLAG peptide, epitopes ofantibodies of interest and the like [A. Yamakawa, Jikken Igaku, 13,469-474 (1995)].

In addition, its production can be increased in accordance with themethod described in Japanese Published Unexamined Patent Application No.227075/90 using a gene amplification system in which a dihydrofolatereductase gene or the like is used.

General methods for isolation and purification of enzymes can be usedfor isolating and purifying the polypeptide of the present inventionfrom a culture of a transformant for use in the production of thepolypeptide of the present invention. For example, when the polypeptideof the present invention is accumulated in a soluble form inside thecells of the transformant for use in the production of the polypeptideof the present invention, the cells in the culture are collected bycentrifugation, the cells are washed and then the cells are disrupted byusing ultrasonic oscillator, French press, Manton Gaulin homogenizer,dynomill or the like to obtain a cell-free extract.

A purified product can be obtained from a supernatant prepared bycentrifuging the cell-free extract, by employing techniques, such assolvent extraction; salting out and desalting with ammonium sulfate orthe like; precipitation with organic solvents; anion exchangechromatography which uses a resin such as diethylaminoethyl(DEAE)-Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Chemical),etc.; cation exchange chromatography in which a resin such asS-Sepharose FF is used (manufactured by Pharmacia), etc.; hydrophobicchromatography in which a resin such as butyl-Sepharose,phenyl-Sepharose, etc is used; gel filtration in which a molecular sieveis used; affinity chromatography; chromatofocusing; electrophoresis suchas isoelectric focusing, etc.; and the like.

Also, when the polypeptide is expressed as an inclusion body in thecells, the cells are recovered, disrupted and centrifuged in the samemanner, the polypeptide is recovered from the thus obtained precipitatedfraction in the usual manner and then the inclusion body of thepolypeptide is solubilized by using a polypeptide denaturing agent. Thepolypeptide is made into normal tertiary structure by diluting ordialyzing the solubilized solution in or against a solution which doesnot contain the polypeptide denaturing agent or contains the polypeptidedenaturing agent but in such a low concentration that the protein is notdenatured, and then its purified product is obtained by the aboveisolation and purification method.

When the polypeptide is secreted extracellurary, the culture is treatedby centrifugation or the like means to obtain a soluble fraction. Apurified preparation of the polypeptide can be obtained from the solublefraction by a method similar to the above method for its isolation andpurification from a cell-free extract supernatant.

Alternatively, the polypeptide can be purified in accordance with thegeneral purification method of glycosyltransferases [Methods inEnzymology, 83, 458].

Also, the polypeptide of the present invention can be purified byproducing it as a fusion protein with other protein and then treatingthe product with affinity chromatography in which a substance havingaffinity for the fused protein is used. For example, the polypeptide ofthe present invention can be purified by producing it as a fusionprotein with protein A and then treating the fusion protein with anaffinity chromatography in which immunoglobulin G is used, in accordancewith the method of Lowe et al. [Proc. Natl. Acad. Sci. USA, 86, 8227(1989), Genes Develop., 4, 1288 (1990)] or the method described inJapanese Published Unexamined Patent Application No. 336963/93 or WO94/23021. Also, the polypeptide of the present invention can be purifiedby producing it as a fusion protein with FLAG peptide and then treatingthe product with an affinity chromatography in which anti-FLAG antibodyis used [Proc. Natl. Acad. Sci. USA, 86, 8227 (1989), Genes Develop., 4,1288 (1990)].

In addition, it can also be purified by affinity chromatography in whichan antibody for the polypeptide itself is used.

Also, the β1,3-N-acetylglucosaminyltransferase of the present inventioncan be produced in accordance with known method [J. Biomolecular NMR, 6,129-134, Science, 242, 1162, J. Biochem., 110, 166 (1991)] by using anin vitro transcription translation system.

In addition, the polypeptide of the present invention can also beproduced by the chemical synthesis method such as Fmoc method(fluorenylmethyloxycarbonyl method), tBoc method (t-butyloxycarbonylmethod) and the like. Also, it can be chemically synthesized using apeptide synthesizing machine manufactured, e.g., by Advanced ChemTech,PERKIN ELMER, Pharmacia Biotech, Protein Technology Instrument,Synthecell-Vega, PerSeptive, Shimadzu or the like.

The purified polypeptide of the present invention can be structurallyanalyzed in the method generally used in protein chemistry, such as themethod described in Protein Structure Analysis for Gene Cloning (editedby H. Hirano, published by Tokyo Kagaku Dojin, 1993).

A β1,3N-acetylglucosaminyltransferase activity of the novel polypeptideof the present invention can be measured in accordance with knownmeasuring methods [J. Biol. Chem., 268, 27118 (1993), J. Biol. Chem.,267, 2994 (1992), J. Biol. Chem., 263, 12461 (1988), Jpn. J. Med. Sci.Biol., 42, 77 (1898)].

A β1,3-galactosyltransferase activity of the polypeptide of the presentinvention can be measured in accordance with known measuring methods [J.Biol. Chem., 2580,9893 (1983), J. Biol. Chem., 262, 15649 (1987), Archi.Biochem. Biophys., 270, 630 (1989), Archi. Biochem. Biophys., 274, 14(1989), Japanese Published Unexamined Patent Application No. 181759/94,J. Biol. Chem., 273, 58 (1998), J. Biol. Chem., 273, 433 (1998), J.Biol. Chem., 273, 12770 (1998), J. Biol. Chem., 274, 12499 (1999)].

(4) Production of a Sugar Chain Having a Structure in whichN-acetylglucosamine is Added to a Galactose Residue Via a β1,3-linkageand Production of a Complex Carbohydrate Containing the Sugar Chain

Sugar chains or complex carbohydrates can be produced by culturing atransformant selected from the transformants derived frommicroorganisms, animal cells, plant cells and insect cells, obtained inthe above (3), in a medium to produce and accumulate a sugar chainhaving a structure in which N-acetylglucosamine is added to a galactoseresidue via a β1,3-linkage or a complex carbohydrate containing thesugar chain, in the culture, and then recovering the sugar chain orcomplex carbohydrate from the culture.

Examples of the structure in which N-acetylglucosamine is added to agalactose residue via a β1,3-linkage include a GlcNAcβ1-3Galβ1-4GlcNAcstructure, a GlcNAcβ1-3Galβ1-3GlcNAc structure, a GlcNAcβ1-3Galβ1-4Glcstructure, a (Galβ1-4GlcNAcβ1-3)_(n)Galβ1-4GlcNAc structure (n≧1), a(Galβ1-4GlcNAcβ1-3)_(n)Galβ1-4Glc structure (n≧1) or the like.

The culturing of the transformants can be carried out in accordance withthe above (3),

Among the above transformants, by simultaneously producing thepolypeptide of the present invention and an recombinant glycoprotein ofinterest (e.g., a recombinant glycoprotein for medical use) in atransformant capable of synthesizing sugar chains, a sugar chain havinga structure in which N-acetylglucosamine is added to a galactose residuevia a β1,3-linkage can be added to the recombinant glycoprotein.

Also, a sugar chain having a structure in which N-acetylglucosamine isadded to a galactose residue via a β1,3-linkage or a complexcarbohydrate to which the sugar chain is added can be produced inaccordance with the method of the above (3) by using an animal or plantobtained in the above (3).

That is, in an animal, a sugar chain having a structure in whichN-acetylglucosamine is added to a galactose residue via a β1,3-linkageor a complex carbohydrate to which the sugar chain is added can beproduced by, for example, rearing a non-human transgenic animalretaining the DNA of the present invention to thereby produce andaccumulate the sugar chain having a structure in whichN-acetylglucosamine is added to a galactose residue via a β1,3-linkageor a complex carbohydrate to which the sugar chain is added, in theanimal, and then recovering the product from the animal.

Examples of the production and accumulation region in the animal includemilk, eggs and the like of the animal.

In a plant, a sugar chain having a structure in whichN-acetylglucosamine is added to a galactose residue via a β1,3-linkageor a complex carbohydrate to which the sugar chain is added can beproduced by, for example, cultivating a transgenic plant having the DNAof the present invention to produce and accumulate the sugar chainhaving a structure in which N-acetylglucosamine is added to a galactoseresidue via a β1,3-linkage or a complex carbohydrate to which the sugarchain is added, in the plant, and then recovering the product from theplant.

A reaction product in which N-acetylglucosamine is added to a galactoseresidue existing at the non-reducing end of a sugar chain or galactosemonosaccharide via a β1,3-linkage can be produced in the followingmethod, in an aqueous medium by using the polypeptide of the presentinvention obtained by the method described in the above (3) as an enzymesource.

That is, the reaction product can be produced by using galactosemonosaccharide, an oligosaccharide having a galactose residue at itsnon-reducing terminal or a complex carbohydrate having a galactoseresidue at the non-reducing terminal of its sugar chain as an acceptorsubstrate and by using the polypeptide of the present invention obtainedby the method described in the above (3) as an enzyme source, byallowing the acceptor substrate, the enzyme source and UDP-GlcNAc to bepresent in an aqueous medium to thereby produce and accumulate areaction product in which N-acetylglucosamine is added to galactose or agalactose residue of the acceptor substrate via a β1,3-linkage, in theaqueous medium, and then recovering the reaction product from theaqueous medium.

When the activity capable of producing 1 μmol ofGlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc within 1 minute at 37° C. usinglacto-N-neotetraose (Galβ1-4GlcNAcβ1-3Galβ1-4Glc) as a substrate isdefined as one unit (U), the enzyme source is used at a concentration of0.1 mU/1 to 10,000 U/l, preferably 1 mU/l to 1,000 U/l.

Examples of the aqueous medium include a buffer such as water,phosphate, carbonate, acetate, borate, citrate, tris, etc.; alcohol suchas methanol, ethanol, etc.; ester such as ethyl acetate, etc.; ketonesuch as acetone, etc.; amide such as acetamide, etc.; and the like. Inaddition, the culture of a transformant obtained by the culturingdescribed in the above (2) or the milk obtained from a non-humantransgenic animal described in the above (2) can also be used as anaqueous medium. If necessary, a surfactant or an organic solvent may beadded to the aqueous medium.

The surfactant may be any agent which can accelerate the production of asugar chain having a structure in which N-acetylglucosamine is added toa galactose residue via a β1,3-linkage or a complex carbohydrate towhich the sugar chain is added. Examples include a nonionic surfactantsuch as polyoxyethylene octadecylamine (e.g., Nymeen S-215, manufacturedby Nippon Oil & Fats), etc.; a cationic surfactant such ascetyltrimethylammonium bromide, alkyldimethyl benzylammoniumchloride(e.g., Cation F2-40E, manufactured by Nippon Oil & Fats), etc.; ananionic surfactant such as lauroyl sarcosinate, etc.; tertiary aminesuch as alkyldimethylamine (e.g., Tertiary Amine FB, manufactured byNippon Oil & Fats), etc.; and the like, which may be used alone or as amixture of two or more. The surfactant is used generally at aconcentration of 0.1 to 50 g/l.

Examples of the organic solvent include xylene, toluene, aliphaticalcohol, acetone, ethyl acetate and the like, which are used generallyat a concentration of 0.1 to 50 ml/l.

Examples of the UDP-GlcNAc include a commercially available preparation,a reaction solution made by using the activity of a microorganism or thelike, and a preparation purified from the reaction solution. UDP-GlcNAccan be used at a concentration of 0.1 to 500 mmol/l.

Examples of the oligosaccharide having a galactose residue at itsnon-reducing terminal include Galβ1-4Glc, Galβ1-4GldNAc,Galβ1-4GlcNAcβ1-3Galβ1-4Glc, Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAc,Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc,Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4GlcNAc,Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc,Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAc, Galβ1-3GlcNAc,Galβ1-3GlcNAcβ1-3Galβ1-4Glc, Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAc,Galβ1-3GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc,Galβ1-3GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAc,Galβ1-4GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-4Glc,Galβ1-4GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-4GlcNAc,Galβ1-4GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4Glc,Galβ1-4GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4GlcNAc,Galβ1-3GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-4Glc,Galβ1-3GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-4GlcNAc,Galβ1-3GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4Glc andGalβ1-3GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4GlcNAc, or an oligosaccharidehaving any one of the structures of these oligosaccharides at thenon-reducing terminal of its sugar chain. Examples of the complexcarbohydrate having a galactose residue at the non-reducing terminal ofits sugar chain include a complex carbohydrate containing a sugar chainhaving any one of the structures of the above oligosaccharides at thenon-reducing terminal of the sugar chain, a complex carbohydratecontaining an asialo complex N-linked sugar chain and the like.

The acceptor substrate can be used at a concentration of 0.01 to 500mmol/l.

If necessary, an inorganic salt such as MnCl₂, etc., β-mercaptoethanol,polyethylene glycol and the like can be added in the productionreaction.

The production reaction is carried out at pH 5 to 10, preferably pH 6 to8, and at 20 to 50° C. for 1 to 96 hours.

A part of a sugar chain can be recovered from the sugar chain or complexcarbohydrate produced by the above method by the known enzymatic methodor the chemical method [Second Series Biochemical Experiment Course,Vol. 4, Methods for Complex Carbohydrate Experiments I, II, edited byJapanese Biochemical Society, Tokyo Kagaku Dojin (1986); GlycobiologyExperiment Protocol, edited by N. Taniguchi, A. Suzuki, K. Furukawa andK. Sugawara, Shujun-sha (1996)].

(5) Use of the DNA or Oligonucleotide of the Present Invention forTreatment, Diagnosis and the Like of Diseases

The DNA of the present invention can be used in the treatment ofdiseases, such as inhibition of inflammation and metastasis, inaccordance with antisense RNA/DNA technique [Bioscience and Industry,50, 322 (1992), Chemistry, 46, 681 (1991), Biotechnology, 9, 358 (1992),Trends in Biotechnology, 10, 87 (1992), Trends in Biotechnology, 10, 152(1992), Cell Engineering, 16, 1463 (1997)] or triple helix technique[Trends in Biotechnology, 10, 132 (1992)].

Also, inflammation and cancer can be diagnosed by measuring theexpression level of the DNA of the present invention in the Northernhybridization method (Molecular Cloning, 2nd Ed), PCR method [PCRProtocols, Academic Press (1990)] or real time PCR method [Jikken Igaku(Supplement), 15, 46 (1997)]. Particularly, quantitative PCR method[Proc. Natl. Acad. Sci. USA, 87, 2725 (1990)] and real time PCR methodare excellent in determination property. For example, production of thepolypeptide of the present invention can be inhibited by administeringthe DNA or oligonucleotide of the present invention or a derivativethereof.

That is, it is possible to inhibit transcription of the DNA encoding thepolypeptide of the present invention or to inhibit translation of mRNAsencoding the polypeptide of the present invention, by using the DNA oroligonucleotide of the present invention or a derivative thereofdescribed in the above (1).

Also, the expression level of the DNA encoding the polypeptide of thepresent invention can be determined in the Northern hybridization methodor PCR method by using the DNA of the present invention or theoligonucleotide prepared from the DNA.

In addition, it is possible to obtain a promoter region of the gene inthe known method [New Cell Engineering Experiment Protocol, edited byAnticancer Study Division, Institute of Medical Science, TokyoUniversity, Shujun-sha (1993)] using the DNA of the present invention asa probe.

Currently, a large number of sequences of function-unknown humanchromosomal genes are registered for data bases. Accordingly, there is apossibility that a human chromosomal gene encoding the polypeptide ofthe present invention can be identified and its structure can be found,by comparing a human cDNA sequence encoding the polypeptide of thepresent invention with sequences of human chromosomal genes registeredfor data bases. When a chromosomal gene sequence identical to the cDNAsequence is registered, promoter region, exon and intron structures ofthe chromosomal gene encoding the polypeptide of the present inventioncan be determined by comparing the cDNA sequence with the chromosomalgene sequence.

Examples of the promoter region include all promoter regions which isinvolved in the transcription of the gene encoding the polypeptide ofthe present invention in mammalian cells. Specific examples includepromoter regions which take part in the transcription of the geneencoding the polypeptide of the present invention in human leukocytes,human colon cancer cells and human pancreatic cancer cells.

It is known that polymorphism and mutation exist in theglycosyltransferase genes. For example, regarding theglycosyltransferase involved in the determination of ABO blood type, thefollowing three kinds of enzymes are formed due to differences in aminoacid sequences based on the genetic polymorphism: an α1,3-N-acetylgalactosamine transferase involved in the synthesis of type A antigen,an α1,3-galactosyltransferase involved in the synthesis of type Bantigen, and an enzyme which has no activity and is involved in theproduction of type O (H) sugar chain [Nature, 345, 229-233 (1990)].

Also, in α1,3-fucosyltransferase (Fuc-T III) which is involved in thedetermination of Lewis blood group, it is known that enzymes whoseactivity is reduced or deleted are formed due to differences in aminoacid sequences based on the gene polymorphism [J. Biol. Chem., 269,29271 (1994), Blood, 82, 2915 (1993), J. Biol. Chem., 269, 20987 (1994),J. Biol. Chem., 272, 21994 (1997)].

It is known that polymorphism of the Fuc-T III gene has a close relationto the expression of, sialyl Lewis a sugar chain which is acancer-related sugar chain antigen in colon cancer [Cancer Res., 56, 330(1996), Cancer Res., 58, 512 (1998)].

Accordingly, it is considered that diagnosis of diseases and predictionof prognosis can be carried out by examining polymorphism of Fuc-T III.

Since the novel β1,3-N-aceatylglucosaminyltransferase of the presentinvention is involved in the synthesis of poly-N-acetyllactosamine, itis considered that it is involved in the synthesis of sialyl Lewis xsugar chain in leukocyte and cancer-related sugar chains (sialyl Lewis xsugar chain, sialyl Lewis a sugar chain, sialyl Lewis c sugar chain,dimeric Lewis a sugar chain) in cancer cells. Accordingly, it isconsidered that diagnosis of inflammation, cancer or metastasis, orprediction of prognosis cancer can be carried out by examining theexpression level and polymorphism of the gene.

Also, it can be used in the diagnosis of other diseases by examining therelationship between the polymorphism of the gene and the diseases inorgans where the gene is expressed.

The polymorphism of the gene can be analyzed by using gene sequenceinformation of the gene. Specifically, the gene polymorphism can beanalyzed in accordance with Southern blot technique, direct sequencingmethod, PCR method, DNA chip method and the like [Rinsho Kensa, 42, 1507(1998), Rinsho Kehsa, 42, 1565(1998)].

(6) Production of an Antibody Capable of Recognizing the Polypeptide ofthe Present Invention

(i) Production of a Polyclonal Antibody

A polyclonal antibody can be produced by using the purified sample ofthe full length or a partial fragment of the polypeptide obtained by themethod of the above (3) or a peptide having an amino acid of a part ofthe protein of the present invention as an antigen and administering itto an animal.

Rabbits, goats, 3- to 20-week-old rats, mice, hamsters and the like canbe used as the animal to be administered. It is preferred that the doseof the antigen is 50 to 100 μg per one animal.

When a peptide is used as an antigen, it is preferred to use the peptideas an antigen after binding it to keyhole limpet haemocyanin, bovinethyroglobulin or the like carrier protein by a covalent bond. Thepeptide to be used as an antigen can be synthesized using a peptidesynthesizer.

The antigen is administered 3 to 10 times at one- to two-week intervalsafter the first administration. Three to seven days after eachadministration, a blood sample is collected from the venous plexus ofthe fundus of the eye, and the serum is tested by enzyme immunoassay[Enzyme-linked Immunosorbent Assay (ELISA): published by Igaku Shoin,(1976), Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory(1988)] and the like as to whether it is reactive with the antigen usedfor immunization.

A polyclonal antibody can be obtained by collecting a serum sample fromnon-human mammal in which the serum showed a sufficient antibody titeragainst the antigen used for immunization, and separating and purifyingthe serum.

Examples of the method for its separation and purification includecentrifugation, salting out with 40 to 50% saturated ammonium sulfate,caprylic acid precipitation [Antibodies—A Laboratory Manual, Cold SpringHarbor Laboratory (1988)] and chromatography using a DEAE-Sepharosecolumn, an anion exchange column, a protein A- or G-column, a gelfiltration column or the like, which may be used alone or incombination.

(ii) Production of a Monoclonal Antibody

(a) Preparation of Antibody Producing Cells

A rat whose serum showed a sufficient antibody titer against the partialfragment of the polypeptide of the present invention used in theimmunization is used as the supply source of antibody producing cells.

Three to seven days after the final administration of the antigensubstance to the rat which showed the antibody titer, the spleen isexcised. The spleen is cut to pieces in MEM medium (manufactured byNissui Pharmaceutical), and cells are unbound using a pair of forcepsand centrifuged at 1,200 rpm for 5 minutes and then the supernatant isdiscarded.

Splenocytes in the thus obtained precipitation fraction are treated witha Tris-ammonium chloride buffer (pH 7.65) for 1 to 0.2 minutes foreliminating erythrocytes and then washed three times with MEM medium,and the thus obtained splenocytes are used as an antibody producingcells.

(b) Preparation of Myeloma Cells

As the myeloma cells, established cell lines obtained from mouse or ratare used.

Examples include 8-azaguanine-resistant mouse (BALB/c-derived) myelomacell lines P3-X63Ag8-U1 (hereinafter referred to as “P3-U1”) [Curr.Topics Microbiol. Immunol., 81, 1 (1978), Eur. J. Immunol., 6, 511(1976)], SP2/O-Ag14(SP-2) [Nature, 276, 269 (1978)], P3-X63-Ag8653 (653)[J. Immunol., 123, 1548 (1979)], P3-X63-Ag8 (X63) [Nature, 256,495(1975)] and the like.

The cell lines are subcultured in an 8-azaguanine medium [prepared bysupplementing RPMI-1640 medium with glutamine (1.5 mmol/l),2-mercaptoethanol (5×10⁻⁵ mol/l), gentamicin (10 μg/ml) and fetal calfserum (FCS) (manufactured by CSL, 10%) and further supplementing theresulting medium (hereinafter referred to as “normal medium”) with8-azaguanine (15 μg/ml)], and they are cultured in the normal medium 3to 4, days before the cell fusion, and 2×10⁷ or more of the cells areused for the cell fusion.

(c) Preparation of a Hybridoma

The antibody producing cells obtained in (a) and the myeloma cellsobtained in (b) are washed thoroughly with MEM medium or PBS (1.83 g ofdisodium hydrogenphosphate, 0.21 g of potassium dihydrogenphosphate and7.65 g of sodium chloride, 1 liter of distilled water, pH 7.2) and mixedin a proportion of antibody producing cells:myeloma cells=5 to 10:1, andthe mixture is centrifuged at 1,200 rpm for 5 minutes and then thesupernatant is discarded.

Cells in the thus obtained precipitation fraction are thoroughlyloosened, and a mixture of 2 g of polyethylene glycol-1000 PEG-1000), 2ml of MEM and 0.7 ml of dimethyl sulfoxide (DMSO) is added to the cellsin an amount of 0.2 to 1 ml per 10⁸ antibody producing cells understirring at 37° C., and then 1 to 2 ml of MEM medium is added severaltimes at 1 to 2 minute intervals. After the addition, the whole volumeis adjusted to 50 ml by adding MEM medium.

After centrifugation of the thus prepared solution at 900 rpm for 5minutes, the supernatant is discarded.

Cells in the thus obtained precipitation fraction are gently loosenedand then suspended in 100 ml of HAT medium [prepared by supplementingthe normal medium with hypoxanthine (10⁻⁴ mol/l), thymidine (1.5×10⁻⁵mol/l) and aminopterin (4×10⁻⁷ mol/l)] by repeated drawing up into ameasuring pipette and discharging from a measuring pipette.

The suspension is dispensed in 100 μl/well portions into a 96-wellculture plate and cultured at 37° C. for 7 to 14 days in a 5% CO₂incubator.

After culturing, a portion of the culture supernatant is taken out andsubjected to an enzyme immunoassay described in Antibodies—A LaboratoryManual, Cold Spring Harbor Laboratory, Chapter 14 (1988) or the like,and hybridomas which specifically react with the partial fragmentpolypeptide of the polypeptide of the present invention are selected.

As an example of the enzyme immunoassay, the following method is shown.

The partial fragment of the polypeptide of the present invention used asthe antigen in carrying out the immunization is coated on an appropriateplate, allowed to react with a first antibody, namely a hybridomaculture supernatant or the purified antibody obtained in the following(d), further allowed to react with a second antibody, namely an anti-rator anti-mouse immunoglobulin antibody labeled with biotin, an enzyme, achemiluminescence substance, a radioactive compound or the like, andthen subjected to the reaction corresponding to the label, and thosewhich react specifically with the polypeptide of the present inventionare selected as hybridomas that produce the monoclonal antibody for thepolypeptide of the present invention.

Using the hybridomas, cloning is repeated twice by limiting dilutionusing HT medium (a medium prepared by eliminating aminopterin from, HATmedium) for the first cloning and the normal medium for the second, andthose in which high antibody titer is constantly observed are selectedas hybridomas that produce an anti-polypeptide antibody for thepolypeptide of the present invention.

(d) Preparation of a Monoclonal Antibody

The hybridoma cells capable of producing a monoclonal antibody for thepolypeptide of the present invention obtained in (c) are injected intothe abdominal cavity of 8 to 10-week old mice or nude mice treated withpristane [by intraperitoneal administration of 0.5 ml of2,6,10,14-tetramethylpentadecane (pristane) followed by 2 weeks offeeding] at a dose of 5 to 20×10⁶ cells per animal. The hybridoma causesascites tumor in 10 to 21 days. The ascitic fluid is collected from theascites tumor-carring mice and centrifuged at 3,000 rpm for 5 minutes toremove the solid matter.

The monoclonal antibody can be obtained by purifying it from the thusobtained supernatant by the same method used in the purification of apolyclonal antibody.

The subclass of the antibody is determined using a mouse monoclonalantibody typing kit or a rat monoclonal antibody typing kit. The amountof the protein is calculated by the Lowry method or from the absorbanceat 280 nm.

(7) Use of the Antibody of the Present Invention

-   (a) The polypeptide of the present invention can be detected by    using the antibody of the present invention. Examples of detection    methods include the ELISA method by using a microtiter plate, the    fluorescent antibody technique, the Western blot technique and the    like.-   (b) The antibody of the present invention can be used in the    immunological staining of cells capable of expressing the    polypeptide of the present invention.-   (c) The antibody of the present invention can be used in the    diagnosis of inflammation and cancer.    (8) Application to Screening Method

Since the novel β1,3-N-acetylglucosaminyltransferase polypeptide of thepresent invention is involved in the synthesis of apoly-N-acetyllactosamine sugar chain, it is possible to increase ordecrease the amount of a poly-N-acetyllactosamine sugar chain which issynthesized in cells, by using a compound capable of increasing orinhibiting the β1,3-N-acetylglucosaminyltrans ferase activity of thepolypeptide.

Also, it is possible to control the amount of a poly-N-acetyllactosaminesugar chain which is synthesized in cells, by controlling the expressionof the polypeptide with a compound capable of accelerating or inhibitingtranscription process of the gene encoding the polypeptide ortranslation process of a protein from the transcription product.

Since it is known that a sialyl Lewis x sugar chain and a sialyl Lewis asugar chain existing on a poly-N-acetyllactosamine sugar chain areligands for selectin, it is considered that a compound capable ofinhibiting the synthesized amount of a poly-N-acetyllactosamine sugarchain is useful for anti-inflammation and metastasis inhibition. On theother hand, it is considered that a compound capable of increasing thesynthesis amount of a poly-N-acetyllactosamine sugar chain is useful forthe production of a poly-N-acetyllactosamine sugar chain and a complexcarbohydrate to which a poly-N-acetyllactosamine sugar chain is added.

Such compounds can be obtained by the methods shown in the following (a)to (e).

-   (a) By using a polypeptide having the novel    β1,3-N-acetylglucosaminyltransferase activity of the present    invention, prepared in accordance with the method described in the    above (3) (a purified preparation, or a cell extract or culture    supernatant of a transformant capable of expressing the    polypeptide), as an enzyme, and in the presence of a test sample,    the β1,3-N-acetylglucosaminyltransferase activity is measured by    known methods [J. Biol. Chem., 268, 27118 (1993), J. Biol. Chem.,    267, 2994 (1992), J. Biol. Chem., 263, 12461 (1988), Jpn. J. Med.    Sci. Biol., 42, 77 (1989)], and a compound which has the activity of    increasing or decreasing the β1,3-N-acetylglucosaminyltransferase    activity is selected and obtained.-   (b) A cell capable of expressing the polypeptide of the present    invention or the transformant described in (3) is cultured for 2    hours to 1 week by the culturing method described in (2) in the    presence of a test sample, and then the amount of a    poly-N-acetyllactosamine sugar chain on the cell surface is measured    by using an antibody (anti-i antibody or anti-I antibody) or a    lectin (LEA, PWM or DSA), which recognizes the sugar chain to    thereby select and obtain a compound which has the activity of    increasing or decreasing the sugar chain content.

Examples of the method for measuring using the antibody or lectininclude detection methods, such as ELISA method by using a microtiterplate, the fluorescent antibody technique, the Western blot technique,the immunological staining or the like can. It can also be measured byusing FACS.

-   (c) A cell capable of expressing the polypeptide of the present    invention is cultured for 2 hours to 1 week by the culturing method    described in (2) in the presence of a test sample, and then amount    of the polypeptide in the cell is measured using the antibody of the    present invention described in (5) to thereby select and obtain a    compound which has the activity of increasing or decreasing the    polypeptide content.

Examples of the measuring method by using the antibody of the presentinvention include detection methods, such as ELISA method by using amicrotiter plate, the fluorescent antibody technique, the Western blottechnique, the immunological staining or the like.

-   (d) A cell capable of expressing the polypeptide of the present    invention is cultured for 2 hours to 1 week by the culturing method    described in (2) in the presence of a test, sample, and then amount    of the transcription product of the gene encoding the polypeptide in    the cell is measured by using a method such as the Northern    hybridization, PCR or the like described in (4) to thereby select    and obtain a compound which has the activity of increasing or    decreasing the transcription product content.-   (e) A plasmid, which contains a DNA consisting of a reporter gene    linked downstream of the promoter obtained in (4), is prepared by    the known method and introduced into an animal cell described in (3)    in accordance with the methods described in (2) and (3) to obtain a    transformant. The transformant is cultured for 2 hours to 1 week by    the culturing method described in (2) in the presence of a test    sample, and then the expression level of the reporter gene in the    cell is measured by known methods [New Cell Engineering Experiment    Protocol, edited by Anticancer Study Division, Institute of Medical    Science, Tokyo University, Shujun-sha (1993), Biotechniques, 20, 914    (1996), J. Antibiotics, 49, 453 (1996), Trends in Biochemical    Sciences, 20, 448 (1995), Cell Engineering, 16, 581 (1997)] to    thereby select and obtain a compound which has the activity of    increasing or decreasing the expression level.

Examples of the reporter gene include a chloramphenicolacetyltransferase gene, a β-galactosidase gene, a β-lactamase gene, aluciferase gene, a green fluorescent protein (GFP) gene and the like.

(9) Production of a Knockout Animal

By using a vector containing the DNA of the present invention, a mutantclone can be produced in which a DNA encoding the polypeptide of thepresent invention, existing on the chromosome in embryonic stem cell ofthe animal of interest such as calf, sheep, goat, pig, horse, domesticfowl, mouse or the like, is inactivated or substituted with an optionalsequence [e.g., Nature, 350, 6315, 243 (1991)], by the known homologousrecombination technique [e.g., Nature, 326, 6110, 295 (1987), Cell, 51,3, 503 (1987) and the like].

By using the thus prepared embryonic stem cell clone, a chimericindividual consisting of the embryonic stem cell clone and normal cellcan be prepared by technique such as the injection chimera method, theaggregation chimera method or the like using blastocyst of an animalfertilized egg. An individual having an mutation of interest in the DNAencoding the polypeptide of the present invention, existing onchromosomes of the whole body cells, can be obtained by the crossing ofthe chimeric individual with normal individual, and a homologousindividual (knockout animal) in which the mutation is induced in both ofthe homologous chromosomes can be obtained by crossing the individuals.

In this way, it is possible to introduce a mutation into a site ofinterest in the DNA encoding the polypeptide of the present inventionexisting on chromosome in an animal individual. For example, byintroducing mutation, such as nucleotide substitution, deletion,insertion or the like, into the translation region of a chromosomal DNAencoding the polypeptide of the present invention, the activity of theproduct can be changed.

Also, it is possible to modify the degree and time of expression, tissuespecificity and the like by introducing similar mutation into theexpression-controlling region. In addition, it is possible to positivelycontrol the expression time, expression region, expression level and thelike by a combination with Cre-loxP system.

As such examples, an example in which a gene of interest was deletedonly in a specified region in the brain using the promoter capable ofexpressing in the region [Cell, 87, 7, 1317 (1996)] and an example inwhich a gene of interest was deleted organ-specifically at a desiredtime by using an adenovirus capable of expressing Cre [Science, 278,5335 (1997)] are known.

Accordingly, regarding the chromosomal DNA encoding the polypeptide ofthe present invention, it is also possible in this manner to prepare ananimal controlling its expression at an optional time in an optionaltissue, or having an optional insertion, deletion or substitution in itstranslation region or expression-controlling region.

In such an animal, symptoms of various diseases caused by thepolypeptide of the present invention can be induced at an optional time,at an optional degree and in an optional region.

Thus, the knockout animal of the present invention becomes an animalmodel markedly useful in the treatment and prevention of variousdiseases caused by the polypeptide of the present invention.Particularly, it is markedly useful as a model for the evaluation oftherapeutic drugs and preventive drugs for such diseases and also offunctional foods, health foods and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing a comparison of the nucleotide sequence ofpositions 1-477 of G4 cDNA with the nucleotide sequence of positions1-451 of G4-2 cDNA.

FIG. 2 is a graph showing a comparison of the nucleotide sequence ofpositions 478-1077 of G4 cDNA with the nucleotide sequence of positions452-1051 of G4-2 cDNA.

FIG. 3 is a graph showing a comparison of the nucleotide sequence ofpositions 1078-1677 of G4 cDNA with the nucleotide sequence of positions1052-1651 of G4-2 cDNA.

FIG. 4 is a graph showing a comparison of the nucleotide sequence ofpositions 1678-2205 of G4 cDNA with the nucleotide sequence of positions1652-2180 of G4-2 cDNA.

FIG. 5 is a dendrogram showing a comparison of the amino acid sequencesof G3, G4, G4-2 and G7 polypeptides, known β1,3-galactosyltransferases(β3Gal-T1, β3Gal-T2, β3Gal-T3, β3Gal-T4, β3Gal-T5) and a knownβ1,3-N-acetylglucosaminyltransferase (β3GnT). Only amino acid sequencesof regions where homology was found are used and compared. That is, theyare compared by excluding cytoplasmic region, membrane-binding regionand stem region.

FIG. 6 is a graph showing construction steps of a plasmid pAMo-G3.

FIG. 7 is a graph showing construction steps of a plasmid pAMo-G4.

FIG. 8 is a graph showing construction steps of a plasmid pAMo-G4-2.

FIG. 9 is a graph showing construction steps of a plasmid pAMo-G7.

FIG. 10 is a result obtained by introducing an expression plasmid(pAMo-G3, pAMo-G4, pAMo-G4-2 or pAMo-G7) and a control plasmid pAMorespectively into Namalwa KJM-1 cell, carrying out indirect fluorescentantibody staining using LEA lectin (thick line), PWM lectin (thick line)or A-PBS (thin line) and then analyzing them using FACS.

FIG. 11 is a graph showing construction steps of a plasmid pAMoF2-G4.

FIG. 12 is a graph showing construction steps of a plasmid pVL1393-F2G4.

FIG. 13 is a graph showing a result obtained by purifying FLAGpeptide-fused secreted G4 from a culture supernatant of Sf21 cellinfected, with a recombinant virus derived from a plasmid pVL1393-F2G4by using Anti-FLAG M1 Affinity gel, and then subjecting it to SDSpolyacrylamide gel electrophoresis (lane 2). As a control, a sample wasprepared from a culture supernatant of Sf21 cell infected with arecombinant virus derived from a plasmid pVL1393 in the same manner andthen subjected to SDS polyacrylamide gel electrophoresis (lane 1). Thearrow shows the position of the produced secreted G4 polypeptide.

FIG. 14 is an electrophoresis pattern showing a result of theexamination of the expression level of G3 transcript in 35 kinds ofhuman organs, using a PCR method. The number of cycles of the PCR is 26.The arrow shows the position of the amplified fragment of target (564bp).

FIG. 15 is an electrophoresis pattern showing a result of theexamination of the expression level of G3 transcript in various humanleukocyte cell lines, human polynuclear leukocyte (PMN), human monocyteand human lymphocyte, using a PCR method. The number of cycles of thePCR is 28. The arrow shows the position of the amplified fragment oftarget (564 bp).

FIG. 16 is an electrophoresis pattern showing a result of theexamination of the expression level of G4 transcript in 35 kinds ofhuman organ, using a PCR method. The number of cycles of the PCR is 26.The arrow shows the position of the amplified fragment of target (202bp).

FIG. 17 is an electrophoresis pattern showing a result of theexamination of the expression level of G4 transcript in various humanleukocyte cell lines, human polynuclear leukocyte (PMN), human monocyteand human lymphocyte, by a PCR method. The number of cycles of the PCRis 28. The arrow shows the position of the amplified fragment of target(202 bp).

FIG. 18 is an electrophoresis pattern showing a result of theexamination of the expression level of G4 transcript in various humancancer cell lines, by a PCR method. The number of cycles of the PCR is27. The arrow shows the position of the amplified fragment of target(202 bp).

FIG. 19 is an electrophoresis pattern showing a result of theexamination of the expression level of G7 transcript in 35 kinds ofhuman organ, by a PCR method. The number of cycles of the PCR is 26. Thearrow shows the position of the amplified fragment of target (456 bp).

FIG. 20 is an electrophoresis pattern showing a result of theexamination of the expression level of G7 transcript in various humanleukocyte cell lines, human polynuclear leukocyte (PMN), human monocyteand human lymphocyte, by a PCR method. The number of cycles of the PCRis 28. The arrow shows the position of the amplified fragment of target(456 bp).

FIG. 21 is a graph showing a result obtained by purifying FLAGpeptide-fused secreted G3 from a culture supernatant of Sf21 cellinfected with a recombinant virus derived from a plasmid pVL1393-F2G3 byusing Anti-FLAG M1 Affinity Gel, and then subjecting it to SDSpolyacrylamide gel electrophoresis (lane 2). As a control, a sample wasprepared from a culture supernatant of Sf21 cell infected with arecombinant virus derived from a plasmid pVL1393 in the same manner andthen subjected to SDS polyacrylamide gel electrophoresis (lane 1). Thearrow shows the position of the produced secreted G3 polypeptide.

FIG. 22 is a graph showing a result obtained by purifying FLAGpeptide-fused secretory G7 from a culture supernatant of Sf21 cellinfected with a recombinant virus derived from a plasmid pVL1393-F2G7 byusing Anti-FLAG M1 Affinity Gel, and then subjecting it to SDSpolyacrylamide gel electrophoresis (lane 2). As a control, a sample wasprepared from a culture supernatant of Sf21 cell infected with arecombinant virus derived from a plasmid pVL1393 in the same manner andthen subjected to SDS polyacrylamide gel electrophoresis (lane 1). Thearrow shows the position considered to be the produced secreted G7polypeptide.

EXPLANATION OF SYMBOLS

-   bp: base pairs-   kb: kilobase pairs-   G418/Km: transposon 5 (Tn5)-derived G418, kanamycin resistance gene-   Ap: pBR322-derived ampicillin resistance gene-   Tc: pBR322-derived tetracycline resistance gene-   P1: pBR322-derived P1 promoter-   Ptk: herpes simplex virus (HSV) thymidine kinase (tk) gene promoter-   Sp. BG: rabbit β-globin gene splicing signal-   A. BG: rabbit β-globin gene poly(A) addition signal-   A. SE: simian virus 40 (SV40) early gene poly(A) addition signal-   A. Atk: poly(A) addition signal of the herpes simplex virus (HSV)    thymidine kinase (tk) gene-   Pmo: long terminal repeat (LTR) promoter of Moloney mouse leukemia    virus-   EBNA-1: EBNA-1 gene of Epstein-Barr virus-   oriP: replication origin of Epstein-Barr virus-   S: gene moiety encoding the signal peptide of immunoglobulin K,-   F: gene moiety encoding FLAG peptide-   G3: DNA (full length or partial length) encoding the    β1,3-N-acetylglucosaminyltransferase G3 obtained by the present    invention-   G4: DNA (full length or partial length) encoding the    β1,3-N-acetylglucosaminyltransferase G4 or    β1,3-N-acetylglucosaminyltransferase G4-2 obtained by the present    invention-   G7: DNA (full length or partial length) encoding the    β1,3-N-acetylglucosaminyltransferase G7 obtained by the present    invention

BEST MODE FOR CARRYING OUT THE INVENTION

Examples are shown below. Unless otherwise indicated, known methodsdescribed in Molecular cloning, 2nd Ed., were used as gene manipulationtechniques.

Example 1 Search for a Candidate Gene Having a Possibility of Encoding aProtein Homologous to a GlcNAc β1,3-N-galactosyltransferase (β3Gal-T1)

The β3Gal-T1 (alias WM1) is a β1,3-galactosyltransferase which isinvolved in the synthesis of Galβ1-3GlcNAc structure (Japanese,Published Unexamined Patent Application No. 181759/94). As a result ofthe search of a gene having homology with the gene of this enzyme or agene possibly encoding a protein having homology with this enzyme at theamino acid level based on gene data bases by using the programs of Blast[J. Mol. Biol., 215 (1990)] and FrameSearch (manufactured by Compugen),several EST (expressed sequence tag) sequences were found. Since theywere classified into three types based on their sequences, it wasconsidered that three kinds of candidate genes are present. Thecandidate genes were named G3, G4 and G7, respectively. As the gene databases, the data base of GenBank and a patented sequence data baseGENESEQ (Derwent, Inc) were used.

An attempt was made to clone fragments of these candidate genes bydesigning primer sets specific for the above three sequences. As theprimer sets, F-3-5 with R-3-5, F-4-5 with R-4-5 and F-7-5a with R-7-3awere used. Sequences of respective primers are shown in SEQ ID NOS:9 to14.

Example 2 Cloning of a Candidate Gene G3

(1) Cloning of a cDNA Fragment for a Candidate Gene G3

By preparing primers specific for the candidate gene G3 (F-3-5 andR-3-5: their sequences are shown in SEQ ID NOS:9 and 10), PCR wascarried out using a single-stranded cDNA prepared from an organ or acell or a cDNA library as the template, and the presence of cDNA havingthe corresponding sequence was examined. As a result, a DNA fragment ofabout 600 bp was amplified when a leukocyte cDNA library (manufacturedby Clontech) or a gastric mucosa cDNA library was used as the template.The specific method is shown below.

The leukocyte cDNA library (phage library: manufactured by Clontech) wasdivided into respective pools of about 40,000 independent clones, andthen. PCR was carried out using phage particles (about 1×10⁷) of eachpool as the template. After 49.5 μl of a reaction solution [10 mmol/lTris-HCl (pH 8.3), 50 mmol/l KCl, 1.5 mmol/l MgCl₂, 0.2 mmol/l dNTP,0.001%(w/v) gelatin and 0.2 μmol/l gene-specific primer] containing thephage particles (about 1×10⁷) which had been treated at 99° C. for 10minutes was heated at 97° C. for 5 minutes, it was cooled on ice for 5minutes. Next, recombinant Taq DNA polymerase (manufactured by TaKaRa)was added thereto, and 30 cycles of the reaction using a reaction systemconstituted by 1 minute at 94° C., 1 minute at 65° C. and 2 minutes at72° C. as 1 cycle were carried out.

The human gastric mucosa cDNA library was produced as follows. A cDNAlibrary was produced by synthesizing cDNA from human gastric mucosapoly(A)⁺ RNA in the cDNA Synthesis System (manufactured by GIBCO BRL),adding an EcoRI-NotI-SalI adapter (Super Choice System for cDNASynthesis; manufactured by GIBCO BRL) to its both termini, inserting itinto the EcoRI site of a cloning vector λZAPII (λZAPII/EcoRI/CIAPCloning Kit, manufactured by STRATAGENE) and then carrying out in vitropackaging by using Gigapack III Gold Packaging Extract manufactured bySTRATAGENE.

The gastric mucosa cDNA library (phage library) was divided intorespective pools of about 50,000 independent clones, and then PCR wascarried out by using phage particles (about 1×10⁷) of each pool as thetemplate. The method was the same as the above method.

The DNA fragment of about 600 bp amplified from a leukocyte cDNA librarywas inserted into a T-vector pT7Blue (manufactured by Novagen) toconstruct a plasmid pT7B-G3FR. As a result of the determination of afull nucleotide sequence of a cDNA fragment contained in pT7B-G3FR, itwas confirmed that the sequence of the cDNA fragment coincided with oneof the EST sequences found in Example 1. For the determination ofnucleotide sequence, a DNA sequencer manufactured by LI-COR (dNAsequencer model 4000L), a DNA sequencer 377 manufactured by PERKIN ELMERand a reaction kit for each sequencer were used.

(2) Cloning of a Full-Length cDNA for the Candidate Gene G3

In order to obtain a full-length cDNA for G3, a digoxigenin-labeledprobe was produced by PCR DIG probe synthesis Kit (manufactured byBoehringer Mannheim). After 39 μl of a reaction solution containing 1 μgof pT7B-G3FR and 0.2 Kmol/l of primers (F-3-5 and R-3-5) was heated at97° C. for 5 minutes, it was cooled on ice for 5 minutes. Next, 1 unitof recombinant Taq DNA polymerase (manufactured by TaKaRa) was addedthereto, and 30 cycles of the reaction using a reaction systemconstituted by 1 minute at 94° C., 1 minute at 65° C. and 1 minute at72° C. as 1 cycle were carried out. A composition of the reactionsolution was as described in the instructions attached to the kit.

A pool of the gastric mucosa cDNA library (about 50,000 independentclones) in which amplification was found in the above (1) was subjectedto plaque hybridization by using the digoxigenin-labeled probe.

A filter on which plaque-derived DNA samples had been transferred wassoaked in 25 ml of a buffer containing 5× concentration of SSPE[composition of 1×SSPE comprises 180 mmol/l sodium chloride, 10 mmol/lsodium dihydrogenphosphate and 1 mmol/l ethylenediaminetetraacetic acid(EDTA) (pH 7.4)], 5× concentration of Denhart's solution [composition of1× Denhart's solution comprises 0.02% (w/v) bovine serum albumin, 0.02%(w/v) Ficoll 400 and 0.02% (w/v) polyvinyl pyrrolidone], 0.5% sodiumdodecyl sulfate (SDS) and 20 μg/ml sermon sperm DNA (hereinafterreferred to as “hybridization buffer”), and pre-hybridization wascarried out at 65° C. for 1 hour.

Next, the filter was soaked in 10 ml of the hybridization buffercontaining 5 μl of the digoxigenin-labeled probe prepared in the above,and hybridization was carried out at 65° C. for 16 hours.

Thereafter, the filter was washed twice under conditions of soaking itin a buffer containing 2×SSPE and 0.1% SDS at 65° C. for 10 minutes,once under conditions of soaking it in a buffer containing 1×SSPE and0.1% SDS at 65° C. for 15 minutes and then twice under conditions ofsoaking it in a buffer containing 0.2×SSPE and 0.1% SDS at 65° C. for 10minutes.

As a result of the plaque hybridization, one hybridized independentclone was obtained. Phage DNA was prepared from this clone by using akit manufactured by Qiagen (QIAGEN Lambda System). The phage DNA wasdigested with XbaI and SalI, and the resulting XbaI-SalI fragment ofabout 1.9 kb was subcloned between XbaI-SalI of pBlue II SK(+). The thusconstructed plasmid was named pBS-G3.

(3) Determination of a Nucleotide Sequence of the cDNA Inserted intoPlasmid pBS-G3

A full nucleotide sequence of the cDNA contained in the pBS-G3 obtainedin the above (2) was determined by the following method.

By using primers (M13-20 primer and reverse primer) specific for asequence in pBlue II SK(+), 5′ side and 3′ side sequences of the cDNAwere determined. Synthetic DNAs specific for the determined sequenceswere produced, and further nucleotide sequences of the cDNA weredetermined by using the DNAs as primers. The full nucleotide sequence ofthe cDNA was determined by repeating this procedure.

For determination of nucleotide sequence, a DNA sequencer manufacturedby LI-COR (dNA sequencer model 4000L) and a reaction kit (SequithermEXCEL II™ Long-Read™ DNA-sequencing kit-Lc: manufactured by AR BROWN),or a DNA sequencer 377 manufactured by PERKIN ELMER and a reaction kit(ABI Prism™ BigDye™ Terminator Cycle Sequencing Ready Reaction kit:manufactured by Applied Biosystems), were used. The full nucleotidesequence (1,912 bp) of the cDNA contained in pBS-G3 is shown in SEQ IDNO:5.

The cDNA encoded a polypeptide containing 397 amino acids havingstructure characteristic of the glycosyltransferases. The polypeptidewas named G3 polypeptide, and its amino acid sequence is shown in SEQ IDNO:1.

The polypeptide showed 19% to 24% homology at amino acid level with thefive human β1,3-galactosyltransferases so far cloned (β3Gal-T1,β3Gal-T2, β3Gal-T3, β3Gal-T4 and β3Gal-T5) [Japanese PublishedUnexamined Patent Application No. 181759/94, J. Biol Chem., 273, 58(1998), J. Biol. Chem., 273, 433 (1998), J. Biol. Chem., 273, 12770(1998), J. Biol. Chem., 274, 12499 (1999)].

The homology analysis was carried out using a Search Homology ofsequence analysis soft GENETYX-MAC 10.1.

Also, the polypeptide showed homology of about 15% at amino acid levelwith the β1,3-N-acetylglucosaminyltransferase (β3GnT) so far cloned[Proc. Natl. Acad. Sci. USA, 96, 406 (1999)], and it was considered thatthe polypeptide comprises an N-terminal cytoplasmic region containing 9amino acids, a subsequent membrane-binding region rich in hydrophobicnature containing 19 amino acids, a stem region containing at least 12amino acids and the remaining C-terminal part comprising most part ofthe polypeptide and containing a catalytic region.

Also, based on the comparison of its amino acid sequence with that ofthe glycosyltransferase having homology and the information on the stemregion and catalytic region of the glycosyltransferase (JapanesePublished Unexamined Patent Application No. 181759/94), it wasconsidered that the stem region comprises at least 12 amino acids.Accordingly, it is considered that the polypeptide having an amino acidsequence of positions 41-397 contains a catalytic region.

Based on these results and the results of Example 8 which will bedescribed later, it was considered that this polypeptide is a novelβ1,3-N-acetylglucosaminyltransferase.

The nucleotide sequence of SEQ ID NO:5 almost coincided with thesequence publicized by WO 98/44112. Also, amino acid sequence of thepolypeptide (SEQ ID NO:1) encoded by this nucleotide sequence coincidedwith the sequence publicized by WO 98/44112.

However, the published patent predicts that the polypeptide is asecretory protein which is clearly a mistake, because it is actually atype II membrane protein. Also, this publication predicts that thepolypeptide is a cardiac and pancreatic protein belonging to theskeletal muscle-derived growth factor super family based on its homologywith other proteins, but it does not reveal its actual activity. Thepublished patent describes general methods for producing the polypeptidein E. coli, insect cells and animal cells, but does not describe data onactual expression of the polypeptide. The present invention found forthe first time that the polypeptide is aβ1,3-N-acetylglucosaminyltransferase and has usefulness for thesynthesis of sugar chains.

Escherichia coli MM294/pBS-G3 as E. coli having pBS-G3 has beendeposited on Apr. 7, 1999, in National Institute of Bioscience and HumanTechnology, Agency of Industrial Science and Technology (postal code305-8566, 1-1-3, Higashi, Tsukuba-shi, Ibaraki, Japan) as FERM BP-6694.

Example 3 Cloning of a Candidate Gene G4

(1) Cloning of a cDNA Fragment for a Candidate Gene G4

Primers specific for the candidate gene G4 (F-4-5 and R-4-5: theirsequences are shown in SEQ ID NOS:11 and 12) were produced, PCR wascarried out by using a single-stranded cDNA prepared from an organ or acell or a cDNA library as the template, and the presence of cDNA havingthe corresponding sequence was examined. As a result, a DNA fragment ofabout 200 bp was amplified when a gastric mucosa cDNA library was usedas the template. A specific method is the same as the method describedin Example 1, except that the primers were changed.

The amplified DNA fragment of about 200 bp was inserted into a T-vectorpT7Blue (manufactured by Novagen) to construct a plasmid pT7B-G4FR. As aresult of the determination, of a full nucleotide sequence of the cDNAfragment contained in pT7B-G4FR, it was confirmed that the sequence ofthe cDNA fragment coincided with one of the EST sequences found inExample 1. For determination of the nucleotide sequence, a DNA sequencermanufactured by LI-COR (dNA sequencer model 4000L), a DNA sequencer 377manufactured by PERKIN ELMER and a reaction kit for each sequencer wereused.

(2) Cloning of a Full-Length cDNA for the Candidate Gene G4

In order, to obtain a full-length cDNA for G4, a digoxigenin-labeledprobe was produced using PCR DIG probe synthesis kit (manufactured byBoehringer Mannheim). After 39 μl of a reaction solution containing 1 μgof pT7B-G4FR and 0.2 μmol/l of primers (F-4-5 and R-4-5) was heated at97° C. for 5 minutes, it was cooled on ice for 5 minutes. Next, 1 unitof recombinant Taq DNA polymerase (manufactured by TaKaRa) was addedthereto, and 30 cycles of the reaction using a reaction systemconstituted by 1 minute at 94° C., 1 minute at 65° C. and 1 minute at72° C. as one cycle were carried out. A composition of the reactionsolution was based on the instructions attached to the kit.

A pool of the gastric mucosa cDNA library (about 50,000 independentclones) in which amplification was found in the above (1) was subjectedto plaque hybridization by using the digoxigenin-labeled probe.

A filter on which plaque-derived DNA samples had been transferred wassoaked in 25 ml of the hybridization buffer, and pre-hybridization wascarried out at 65° C. for 1 hour. Next, the filter was soaked in 10 mlof the hybridization buffer containing 5 μl of the digoxigenin-labeledprobe produced in the above and hybridization was carried out at 65° C.for 16 hours. Thereafter, the filter was washed twice under conditionsof soaking it in a buffer containing 2×SSPE and 0.1% SDS at 65° C. for10 minutes, once under conditions of soaking it in a buffer containing1×SSPE and 0.1% SDS at 65° C. for 15 minutes and then twice underconditions of soaking it in a buffer containing 0.2×SSPE and 0.1% SDS at65° C. for 10 minutes.

As a result of the plaque hybridization, one hybridized independentclone was obtained. By carrying out in vivo excision in accordance witha manual provided by Stratagene, a plasmid pBS-G4-2 was recovered fromthe clone.

In the same manner, a human placenta cDNA library was prepared, and aplasmid pBS-G4 was obtained from the library.

(3) Determination of Nucleotide Sequences of the cDNAs Inserted intoPlasmids pBS-G4 and pBS-G4-2

Full nucleotide sequences of the cDNAs molecules contained in the pBS-G4and pBS-G4-2 obtained in the above (2) was determined by the followingmethod.

By using primers (M13-20 primer and reverse primer) specific for asequence in pBlue II SK(−), 5′ side and 3′ side sequences of the cDNAswere determined. Synthetic DNAs specific for the determined sequenceswere produced, and further nucleotide sequences of the cDNAs weredetermined by using the DNAs as primers. Full nucleotide sequences ofthe cDNAs were determined by repeating this procedure.

For determination of the nucleotide sequence, a DNA sequencermanufactured by LI-COR (dNA sequencer model 4000L) and a reaction kit(Sequitherm EXCEL II™ Long-Read™ DNA-sequencing kit-Lc: manufactured byAR BROWN), or a DNA sequencer 377 manufactured by PERKIN ELMER and areaction kit (ABI Prism™ BigDye™ Terminator Cycle Sequencing ReadyReaction, kit: manufactured by Applied Biosystems), were used.

A full nucleotide sequence (2,205 bp) of the cDNA contained in pBS-G4 isshown in SEQ ID NO:6. The cDNA encoded a polypeptide composed of 372amino acids having a structure characteristic of theglycosyltransferase. The polypeptide was named G4 polypeptide, and itsamino acid sequence is shown in SEQ ID NO:2.

A full nucleotide sequence (2,180 bp) of the cDNA contained in pBS-G4-2is shown in SEQ ID NO:7. The cDNA encoded a polypeptide containing 372amino acids having a structure characteristic of theglycosyltransferase. The polypeptide was named G4-2 polypeptide, and itsamino acid sequence is shown in SEQ ID NO:3.

The cDNA contained in pBS-G4 was named G4 cDNA, and the cDNA containedin pBS-G4-2 was named G4-2 cDNA.

The nucleotide sequence of the 5′ non-translation region was differentbetween G4 cDNA and G4-2 cDNA, and substitution of at least twonucleotides was found (cf. FIGS. 1 to 4). In the translation region, the1111th nucleotide of G4 cDNA was adenine, but the correspondingnucleotide in G4-2 cDNA was substituted with guanine. Accordingly, the328th amino acid of G4 polypeptide is His, while the 328th amino acid ofG4-2 polypeptide is substituted with Arg.

The fact that the nucleotide sequence of the 51 non-translation regionis different between G4 cDNA and G4-2 cDNA indicates that differentpromoters are acting in the placenta and stomach. It is considered thatother nucleotide substitutions are caused by individual difference,somatic cell mutation or error of reverse transcriptase. As shown in thefollowing Example, both of the proteins encoded by G4 cDNA and G4-2 cDNApossessed a β1,3-N-acetylglucosaminyltransferase activity.

Each of the G4 and G4-2 polypeptides showed 22% to 26% homology at aminoacid level with the five human β1,3-galactosyltransferases so far cloned(β3Gal-T1, β3Gal-T2, β3Gal-T3, β3Gal-T4 and β3Gal-T5) [JapanesePublished Unexamined Patent Application No. 181759/94, J. Biol. Chem.,273, 58 (1998), J. Biol. Chem., 273, 433 (1998), J. Biol. Chem., 273,12770 (1998), J. Biol. Chem., 274, 12499 (1999)]. The homology analysiswas carried out by using a Search Homology of sequence analysis softGENETYX-MAC 10.1.

Also, each of these polypeptides showed homology of about 17.5% at aminoacid level with the β1,3-N-acetylglucosaminyltransferase (β3GnT) so farcloned [Proc. Natl. Acad. Sci. USA, 96, 406 (1999)]. It was consideredthat each of the polypeptides comprises an N-terminal cytoplasmic regioncontaining 11 amino acids, a subsequent membrane-binding region rich inhydrophobic nature containing 21 amino acids, a stem region containingat least 12 amino acids and the remaining C-terminal pert comprisingmost of the polypeptide and containing a catalytic region. Based on thecomparison of their amino acid sequences with that of theglycosyltransferase having homology and the information on the stemregion and catalytic region of the glycosyltransferase (JapanesePublished Unexamined Patent Application No. 181759/94), it wasconsidered that the stem region comprises at least 12 amino acids.Accordingly, it is considered that the polypeptide having an amino acidsequence of positions 45-372 contains a catalytic region.

Based on these results and the results of Examples 8, 9, 10 and 12 whichwill be described later, it was considered that each of thesepolypeptides is a novel β1,3-N-acetylglucosaminyltransferase.

The nucleotide sequence of SEQ ID NO:6 or 7 almost coincided with thesequence publicized by WO 98/44112. Also, amino acid sequence of thepolypeptide (SEQ ID NO:2) encoded by the nucleotide sequence of SEQ IDNO:6 coincided with the sequence published by WO 98/21328. However, thepublished patent merely describes that the polypeptide is a type IImembrane protein, but does not reveal actual activity of thepolypeptide. The present invention found for the first time that thepolypeptide is a β1,3-N-acetylglucosaminyltransferase and has usefulnessfor the synthesis of sugar chains.

Escherichia coli MM294/pBS-G4 as E. coli having pBS-G4-2 has beendeposited on Apr. 7, 1999, in National Institute of Bioscience and HumanTechnology, Agency of Industrial Science and Technology (postal code305-8566, 1-1-3, Higashi, Tsukuba-shi, Ibaraki, Japan) as FERM BP-6695.

Example 4 Cloning of a Candidate Gene G7

(1) Cloning of a cDNA for a Candidate Gene G7

Primers specific for the candidate gene G7 (F-7-5a and R-7-3a: theirsequences are shown in SEQ ID NOS:13 and 14) were produced, PCR wascarried out by using a single-stranded cDNA prepared from various organsor cells, or various cDNA libraries as the template, and the presence ofcDNA having the corresponding sequence was examined. As a result, a DNAfragment of about 1.3 kb was amplified when a single-stranded cDNAderived from a human neuroblastoma cell line SK-N-MC was used as thetemplate. The specific method is shown below.

The cell line SK-N-MC was obtained from American Type Culture Collection(ATCC). Total RNA was prepared from the SK-N-MC cells in accordance witha general method [Biochemistry, 18, 5294 (1977)]. A single-stranded cDNAwas synthesized from 5 μg of the total RNA using a kit (SUPERSCRIPT™Preamplification System; manufactured by BRL). The reaction was carriedout in 21 μl, and the solution after the reaction was diluted 50 timeswith water and stored at −80° C. until its use.

After 40 μl of a reaction solution [10 mmol/l Tris-HCl (pH 8.3), 50mmol/l KCl, 1.5 mmol/l MgCl₂, 0.2 mmol/l dNTP, 0.001% (w/v) gelatin and0.2 μmol/l gene-specific primer] containing 10 μl of the single-strandedcDNA was heated at 97° C. for 5 minutes, it was cooled on ice for 5minutes. Next, 1 unit of recombinant Taq DNA polymerase (manufactured byTaKaRa) was added thereto, and 30 cycles of the reaction by using areaction system constituted by 30 seconds at 94° C., 1 minute at 60° C.and 2 minutes at 72° C. as 1 cycle were carried out.

The thus amplified DNA fragment of about 1.3 kb was inserted into aT-vector pT7Blue (manufactured by Novagen) to construct a plasmidpT7B-G7.

(2) Determination of a Nucleotide Sequence of the cDNA Inserted intoPlasmid pT7B-G7

The full nucleotide sequence of the cDNA contained in pT7B-G7 obtainedin the above (1) was determined by the following method.

By using primers (M13-20 primer and reverse primer) specific for asequence in pT7Blue, 5′ side and 3′ side sequences of the cDNA weredetermined. Synthetic DNAs specific for the determined sequences wereproduced, and further nucleotide sequences of the cDNA were determinedusing the DNAs as primers. The full nucleotide sequence of the cDNA wasdetermined by repeating this procedure.

For determination of the nucleotide sequence, a DNA sequencermanufactured by LI-COR (dNA sequencer model 4000L) and a reaction kit(Sequitherm EXCEL II™ Long-Read™ DNA-sequencing kit-Lc: manufactured byAR BROWN), or a DNA sequencer 377 manufactured by PERKIN ELMER and areaction kit (ABI Prism™ BigDye™ Terminator Cycle Sequencing ReadyReaction kit: manufactured by Applied Biosystems), were used.

The full nucleotide sequence (1,296 bp) of the cDNA contained in pT7B-G7is shown in SEQ ID NO:8.

The cDNA encoded a polypeptide composed of 378 amino acids having astructure characteristic of the glycosyltransferase. The polypeptide wasnamed G7 polypeptide, and its amino acid sequence is shown in SEQ IDNO:4.

The G7 polypeptide showed 22% to 25% homology at amino acid level withthe five human β1,3-galactosyltransferases so far cloned (β3Gal-T1,β3Gal-T2, β3Gal-T3, β3Gal-T4 and β3Gal-T5) [Japanese PublishedUnexamined Patent Application No. 181759/94, J. Biol. Chem., 273, 58(1998), J. Biol. Chem., 273, 433 (1998), J. Biol. Chem., 273, 12770(1998), J. Biol. Chem., 274, 12499 (1999)].

The homology analysis was carried out by using a Search Homology ofsequence analysis soft GENETYX-MAC 10.1.

Also, the polypeptide showed homology of about 14.8% at amino acid levelwith the human β1,3-N-acetylglucosaminyltransferase (β3GnT) so farcloned [Proc. Natl. Acad. Sci. USA, 96, 406 (1999)], and it wasconsidered that it comprises an N-terminal cytoplasmic region containing29 amino acids, a subsequent membrane-binding region rich in hydrophobicnature containing 20 amino acids, a stem region containing at least 12amino acids and the remaining C-terminal pert comprising most of thepolypeptide and containing a catalytic region. Based on the comparisonof its amino acid sequence with that of the glycosyltransferase havinghomology and the information on the stem region and catalytic region ofthe glycosyltransferase (Japanese Published Unexamined PatentApplication No. 181759/94), it was considered that the stem regioncomprises at least 12 amino acids. Accordingly, it is considered thatthe polypeptide having an amino acid sequence of positions 62-378contains a catalytic region.

Based on these results and the results of Examples 8 which will bedescribed later, it was considered that the polypeptide is a novelβ1,3-N-acetylglucosaminyltransferase.

Escherichia coli MM294/pT7B-G7 as E. coli having pT7B-G7 has beendeposited on Apr. 7, 1999, in National Institute of Bioscience and HumanTechnology, Agency of Industrial Science and Technology (postal code305-8566, 1-1-3, Higashi, Tsukuba-shi, Ibaraki, Japan) as FERM BP-6696.

Example 5 Homology Analysis on Amino Acid Sequences

A dendrogram was created using the amino acid sequences of G3, G4-2 andG7 polypeptides, amino acid sequences of known humanβ1,3-galactosyltransferases (β3Gal-T1, β3Gal-T2, β3Gal-T3, β3Gal-T4 andβ3Gal-T5) and the amino acid sequence of known humanβ1,3-N-acetylglucosaminyltransferase (β3GnT) (cf. FIG. 5). Thedendrogram was created using CLUSTAL X Multiple Sequence AlignmentProgram (ftp://ftp-iqbmc.u-strasbq.fr/pub/ClustalX/). As a result, itwas found that the G3, G4-2 and G7 polypeptides form one subgroup. TheG3 polypeptide showed 39.6% and 44.5% of homology with G4-2 and G7polypeptides, respectively. The G4-2 polypeptide showed 42.5% homologywith the G7 polypeptide.

EXAMPLE 6 Construction of Expression Plasmids for Animal Cell

In order to express each polypeptide encoded by the G3, G4, G4-2 and G7cDNAs obtained in Examples 2 to 4, expression plasmids were constructedby inserting each cDNA into an expression vector pAMo [J. Biol. Chem.,268, 22782 (1993), alias pAMoPRC3Sc (Japanese Published UnexaminedPatent Application No. 336963/93)].

(1) Construction of Plasmid pAMo-G3 for Expressing G3 Polypeptide (cf.FIG. 6)

pBS-G3 was digested with restriction enzymes XbaI and SalI and thentreated with DNA polymerase Klenow fragment to form blunt ends.Thereafter, SfiI linkers (SEQ ID NOS:15 and 16) were added thereto toobtain an SfiI fragment of about 1.9 kb. On the other hand, pAMo wasdigested with SfiI and BamHI and then an SfiI fragment of 8.7 kb wasobtained. By linking these 2 fragments, an expression plasmid pAMo-G3was constructed.

Synthesis and phosphorylation of the SfiI linkers (SEQ ID NOS:15 and 16)were carried out in accordance with a general method (Japanese PublishedUnexamined Patent Application No. 336963/93).

(2) Construction of Plasmid pAMo-G4 for Expressing G4 Polypeptide (cf.FIG. 7)

pBS-G4 was digested with restriction enzymes HindIII and BstEII and thena HindIII-BstEII fragment of 0.4 kb was obtained. Also, pT7B-G4sec wasdigested with restriction enzymes BstEII and NotI and then a BstEII-NotIfragment of 0.9 kb was obtained. The pT7B-G4sec was constructed by themethod shown in Example 9(1) which will be described later. On the otherhand, pAMo was digested with HindIII and NotI and then a HindIII-NotIfragment of 8.7 kb was obtained. By linking these 3 fragments, anexpression plasmid pAMo-G4 was constructed.

(3) Construction of Plasmid pAMo-G4-2 for Expressing G4-2 Polypeptide(cf. FIG. 8)

pBS-G4-2 was digested with restriction enzymes HindIII and BstEII andthen a HindIII-BstEII fragment of 0.4 kb was obtained. Also, pBS-G4-2was digested with restriction enzymes BstEII and NotI and then aBstEII-NotI fragment of 1.5 kb was obtained. On the other hand, pAMo wasdigested with HindIII and NotI and then a HindIII-NotI fragment of 8.7kb was obtained. By linking these 3 fragments, an expression plasmidpAMo-G4-2 was constructed.

(4) Construction of Plasmid pAMo-G7 for Expressing G7 Polypeptide (cf.FIG. 9)

pT7B-G7 was digested with restriction enzymes SmaI and HincII and thenSfiI linkers (SEQ ID NOs:15 and 16) were added thereto to obtain an SfiIfragment of about 1.3 kb. On the other hand, pAMo was digested with SfiIand BamHI and then an SfiI fragment of 8.7 kb was obtained. By linkingthese 2 fragments, an expression plasmid pAMo-G7 was constructed.

Example 7 Synthesis of poly-N-acetyllactosamine Sugar Chain in CulturedHuman Cells Transformed by a Plasmid Expressing Each Polypeptide of G3,G4, G4-2 and G7

(1) Acquisition of Stable Transformants

Each of a control plasmid (pAMo) and various expression plasmidsconstructed in Example 6 (pAMo-G3, pAMo-G4, pAMo-G4-2 and pAMo-G7) wasdissolved in a buffer containing 10 mmol/l Tris-HCl (pH 8.0) and 1mmol/l EDTA (sodium ethylenediaminetetraacetate) (hereinafter referredto as “TE buffer”) to give a concentration of 1 μl/μl and thenintroduced into Namalwa KJM-1 cell [Cytotechnology, 1, 151 (1988)] byelectroporation method [Cytotechnology, 3, 133 (1990)] to obtainrespective transformants.

After introducing 4 μg per 1.6×10⁶ cells of each plasmid, the cells weresuspended in 8 ml of RPMI 1640/ITPSG medium [RPMI 1640 medium(manufactured by Nissui Pharmaceutical) supplemented with 1/40 volume of7.5% NaHCO₃, 3% of 200 mmol/l L-glutamine solution (manufactured byGIBCO), 0.5% penicillin/streptomycin solution (manufactured by GIBCO,5,000 units/ml penicillin and 5,000 μg/ml streptomycin),N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid(N-2-hydroxyethylpiperazine-N′-2-hydroxypropane-3-sulfonic acid; HEPES)(10 mmol/l), insulin (3 μg/ml), transferrin (5 μl/ml), sodium pyruvate(5 mmol/l), sodium selenite (125 nmol/l) and galactose (1 mg/m/l)] andcultured at 37° C. for 24 hours in a CO₂ incubator. Thereafter, G418(manufactured by GIBCO) was added thereto to give a concentration of 0.5mg/ml, followed by culturing for 14 days to obtain a stabletransformant. Each of the thus obtained transformants was subcultured inRPMI 1640/ITPSG medium containing 0.5 mg/ml of G418.

(2) Measurement of the Amount of poly-N-acetyllactosamine Sugar ChainsExpressed in Each Transformant

The amount of poly-N-acetyllactosamine sugar chains expressed in eachtransformant can be analyzed using FACS after fluorescence staining byusing antibodies or lectins capable of recognizingpoly-N-acetyllactosamine sugar chains. Anti-i antibody (Den) can be usedas an antibody which recognize poly-N-acetyllactosamine sugar chain. Asthe lectin which recognizes poly-N-acetyllactosamine sugar chain, LEA,PWM and DSA can be used.

An example of the method in which a lectin (LEA or PWM) capable ofrecognizing a poly-N-acetyllactosamine sugar chain is used is shownbelow.

The transformed cells (5×10⁶ cells for each transformant) were suspendedin 100 μl of PBS (8 g/l NaCl, 0.2 g/l KCl, 1.15 g/l Na₂HPO₄ (anhydrous)and 0.2 g/l KH₂PO₄) containing 20 mU of Clostridium perfringensneuramimidase (N 2133 manufactured by SIGMA), followed by reacting at37° C. for 1-hour to thereby carrying out the sialidase treatment of thetransformed cells.

The cells (about 1×10⁶) were transferred into a microtube (1.5 ml:manufactured by Eppendorf) and centrifuged (550×g for 7 minutes) tocollect the cells.

The cells were washed with 0.9 ml of a 0.1% sodium azide-containingphosphate buffer PBS (A-PBS: 8 g/l NaCl, 0.2 g/l KCl 1.15 g/l Na₂HPO₄(anhydrous), 0.2 g/l KH₂PO₄, 0.1% sodium azide), and then 20 μl ofFITC-labeled LEA (manufactured by EY Laboratories) diluted to 10 μg/mlwith A-PBS or FITC-labeled PWM (manufactured by EY Laboratories) dilutedto 100 μg/ml with A-PBS was added to the washed cells and suspended,subsequently carrying out the reaction at 4° C. for 1 hour.

After the reaction, the cells were washed once with 0.9 ml of A-PBS andthen suspended in 0.6 ml of A-PBS to carry out the FACS (fluorescenceactivated cell sorter) analysis using FACSCaliber (manufactured byBecton Dickinson Immunocytometry Systems USA). As a control test, thesame analysis was carried out by using A-PBS instead of the lectins.

In the cells into which pAMo-G3, pAMo-G4, pAMo-G4-2 or pAMo-G7 wasintroduced, reactivity for LEA was higher in comparison with thepAMo-introduced, cells (FIG. 10). Also, in the cells into which pAMo-G3,pAMo-G4, pAMo-G4-2 or pAMo-G7 was introduced, reactivity for PWM washigher in comparison with the pAMo-introduced (FIG. 10).

The results show that poly-N-acetyllactosamine sugar chains were newlysynthesized on sugar chains of cell surface glycoproteins or glycolipidsby expressing cDNA of G3, G4, G4-2 or G7 in Namalwa KJM-1 cell.

Also, it shows that poly-N-acetyllactosamine sugar chains were newlysynthesized on sugar chains of glycoproteins or oligosaccharidessecreted from cells in which cDNA of G3, G4, G4-2 or G7 is expressed.Accordingly, it is possible to add a sugar chain containing apoly-N-acetyllactosamine to a glycoprotein produced as a secreted formby secreting and producing a useful glycoprotein by use of cells inwhich cDNA of G3, G4, G4-2 or G7 is expressed as a host.

On the other hand, when the fluorescence staining was carried out on thetransformed cells by using DU-PAN-2 which is an antibody for sialylLewis c sugar chains, reactivity of the antibody was not changed. Themethod was carried out in accordance with a general method [J. Biol.Chem., 274, 12499 (1999)].

Example 8 Measurement of β1,3-N-acetylglucosaminyltransferase Activityin Cultured Human Cells Transformed by a Plasmid Capable of ExpressingG3, G4, G4-2 or G7 polypeptide

The β1,3-N-acetylglucosaminyltransferase activity was examined by usinga cell extract of stable transformants which were transformed by aplasmid capable of expressing G3, G4, G4-2 or G7 polypeptide obtained inExample 7.

The transformed cells (about 2×10⁷) were transferred into a microtube(1.5 ml: manufactured by Eppendorf) and centrifuged (550×g for 7minutes) to collect the cells. The cells were washed with 0.9 ml of PBS,the washed cells were suspended in a solution (100 μl) composed of 20mmol/l HEPES (pH 7.2) and 1% Triton X-100 and then the cells weredisrupted by a sonicator (Bioruptor; manufactured by Cosmo Bio). Afterstanding at 4° C. for 1 hour, a supernatant was obtained bycentrifugation (550×g for 7 minutes). The supernatant was used as anenzyme sample. The β1,3-N-acetylglucosaminyltransferase activity wasmeasured by using this enzyme sample.

Preparation of a pyridylaminated sugar chain substrates and activitymeasurement were carried out in accordance with known methods [JapanesePublished Unexamined Patent Application No. 181757/94, JapanesePublished Unexamined Patent Application No. 823021/94, J. Biol. Chem.,269, 14730 (1994), J. Biol. Chem., 267, 2994 (1992)].

Specifically, the reaction was carried out at 37° C. for 16 hours in 30μl of an assay solution [200 mmol/l of MOPS (pH 7.5), 50 mmol/l ofUDP-GlcNAc (SIGMA), 20 mmol/l of MnCl₂, 0.3% Triton X-100, 50 μM of apyridylaminated sugar chain substrate and 10 μl of the above celllysate], and then products were detected by a high performance liquidchromatography (HPLC).

As a substrate, lacto-N-neotetraose (Galβ1-4GlcNAcβ1-3Galβ1-4Glc;hereinafter referred to as “LNnT”) fluorescence-labeled withaminopyridine was used.

LNnT was purchased from Oxford Glycosystems. Fluorescence labeling ofthe oligosaccharide was carried out in accordance with the generalmethod [Agric. Biol. Chem., 54, 2169 (1990)].

After carrying out the reaction by using the assay solution with orwithout UDP-GlcNAc (sugar donor), HPLC was carried out and peaksappeared only in the assay solution containing UDP-GlcNAc were definedas products.

The assay solution after completion of the reaction was treated at 100°C. for 5 minutes and then centrifuged at 10,000×g for 5 minutes, and aportion (5 μl) of the thus obtained supernatant was applied to HPLC.

The HPLC was carried out by using TSK-gel ODS-80Ts column (4.6×300 mm;Tosoh); 0.02 M ammonium acetate buffer (pH 4.0) was used as an eluate atelution temperature 50° C. and flow rate 0.5 ml/min.

Products were detected by using a fluorescence spectrum photometerFP-920 (JASCO Corporation) (excitation wavelength 320 nm, radiationwavelength 400 nm). In identifying the product, coincidence of itselution time with that of a standard sugar chain was used as the index.As a standard sugar chain, aminopyridylatedGlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc was used.

The amount of products was measured by using pyridylaminated lactose asa standard and comparing their fluorescence intensity.

As a result of the activity measurement by using a cell extract ofstable transfomants which were introduced with a control plasmid (pAMo)or each expression plasmid (pAMo-G3, pAMo-G4, pAMo-G4-2 or pAMo-G7),ratio of a substrate (LNnT) converted into a product(GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc) in cells transformed by anexpression plasmid for G3, G4, G4-2 or G7 polypeptide was increased tobe 2.7%, 2.8%, 2.8% and 2.3% respectively, while it was 1.8% in thecontrol plasmid-introduced cells. That is, it was found that theβ1,3-N-acetylglucosaminyltransferase activity was higher in the cellstransformed by an expression plasmid for G3, G4, G4-2 or G7 polypeptidein comparison with the control plasmid-introduced cells.

Based on these results, it was confirmed that the G3, G4, G4-2 or G7polypeptide is a novel β1,3-N-acetylglucosaminyltransferase. The resultshows a possibility that a sugar chain in which N-acetylglucosamine isadded via a β1,3-linkage to the galactose residue existing at thenon-reducing terminal of a sugar chain can be synthesized by using theG3, G4, G4-2 or G7 polypeptide.

Example 9 Secretion Production of FLAG Peptide-Fusedβ1,3-N-acetylglucosaminyltransferase (G4) by Using Namalwa KJM-1 Cell asa Host

(1) Construction of a FLAG Peptide-Fused Secretion Vector pAMoF2

A secretion vector pAMoF2 was constructed for expressing a protein as asecreted form fused with a FLAG peptide (SEQ ID NO:17) at N-terminal ofthe protein. DNAs encoding a signal sequence of an immunoglobulin κ andthe FLAG peptide were constructed by using 6 synthetic DNA fragments.

A HindIII-Asp718 fragment of about 8.7 kb was obtained by digesting pAMowith HindIII and Asp718. Six DNA fragments [IgK-1 (SEQ ID NO:18), IgK-2(SEQ ID NO:19), IgK-3 (SEQ ID NO:20), IgK-4 (SEQ ID NO:21), IgK-5 (SEQID NO:22) and IgK-6 (SEQ ID NO:23)] were synthesized as linkers forlinking the HindIII-digestion site and Asp718-digested site. Also, eachof the restriction enzyme digestion sites of PmaCI, StuI and SnaBI wasgenerated in the linkers constructed by these DNA fragments. The 6 DNAfragments were respectively synthesized by 380A DNA Synthesizermanufactured by Applied Biosystems. The thus synthesized DNA fragmentswere used after phosphorylation by using T4 polynucleotide ki nase(manufactured by TaKaRa, the same shall apply hereinafter).

A plasmid pAMoF2 was constructed by linking the thus obtained 6phosphorylated synthetic DNA fragments and the HindIII-Asp718 fragmentof about 8.7 kb.

(2) Construction of a Plasmid pAMoF2-i52S

DNA shown by SEQ ID NO:24 (hereinafter referred to as “C12-7”) and DNAshown by SEQ ID NO:25 (hereinafter referred to as “C12-9”) weresynthesized as primers for PCR (they can also be purchased from SawadayTechnology).

They are designed such that a BamHI site is generated in C12-7, and aNotI site in C12-9.

PCR was carried out by using a kit manufactured by TaKaRa (GeneAmp™ DNAAmplification Reagent Kit with AmpliTaq™ Recombinant Taq DNAPolymerase). A reaction solution was prepared in accordance with themethod of the kit, and 10 cycles of a reaction at 94° C. for 30 seconds,65° C. for 1 minute and 72° C. for 0.2 minutes, and subsequent reactionat 72° C. for 7 minutes were carried out by using a DNA thermal cycler(PERKIN ELMER CETUS DNA Thermal Cycler; available from TaKaRa). As atemplate, 10 ng of a plasmid pAMo-i [Proc. Natl. Acad. Sci. USA, 94,14294 (1997)] was used. A DNA fragment of about 1.1 kb was obtained bythe PCR.

A plasmid pT7B-i52S No. 3 was constructed by linking the PCR-amplifiedDNA fragment of about 1.1 kb and a T-vector pT7Blue (manufactured byNovagen).

Next, a plasmid pAMoF2-i52S was constructed.

A StuI-BanIII fragment of about 7.2 kb was obtained by digesting pAMoF2with StuI and BanIII. A BanIII-NotI fragment of about 1.7 kb wasobtained by digesting pAMo with BanIII and NotI. After digestingpT7B-i52S No. 3 with BamHI, the 5′ cohesive end formed by the BamHIdigestion was changed to blunt end using E. coli DNA polymerase I Kleowfragment and then the fragment was digested with NotI to thereby obtaina BamHI(blunt end)-NotI fragment of about 1.1 kb.

The plasmid pAMoF2-i52S was constructed by linking the thus obtainedStuI-BanIII fragment of about 7.2 kb, BanIII-NotI fragment of about 1.7kb and BamHI(blunt end)-NotI fragment of about 1.1 kb.

(3) Construction of a Plasmid pAMoF2-G4 for Secreted Expression of FLAGPeptide-Fused G4 Polypeptide (cf. FIG. 11)

It was considered based on its primary sequence that the clonedβ1,3-N-acetylglucosaminyltransferase (G4) comprises an N-terminalcytoplasmic region containing 11 amino acids, a subsequentmembrane-binding region rich in hydrophobic nature containing 21 aminoacids, a stem region containing at least 12 amino acids and theremaining C-terminal pert comprising most of the polypeptide containinga catalytic region.

Accordingly, secreted expression of the G4 polypeptide was attempted byremoving the N-terminal cytoplasmic, region containing 11 amino acids,the membrane-binding region containing 21 amino acids and a part of thestem region (5 amino acids), and adding an immunoglobulin signalsequence and the FLAG peptide to the removed regions.

First, a plasmid pT7B-G4sec was constructed by preparing a DNA regionencoding a region considered to have the catalytic activity of G4polypeptide (from the 38th glutamic acid to the 372nd tyrosine in SEQ IDNO:2) by PCR and then inserting it into a T-vector pT7Blue (manufacturedby Novagen). A specific method is described below.

G4-SF and G4-SR (their sequences are shown in SEQ ID NOs:26 and 27) weresynthesized as primers for PCR (they can also be purchased from SawadayTechnology).

PCR was carried out using a kit manufactured by TaKaRa (GeneAmp™ DNAAmplification Reagent Kit with AmpliTaq™ Recombinant Taq-DNAPolymerase). The reaction solution was prepared in accordance with themethod of the kit, and 10 cycles of a reaction at 94° C. for 30 seconds,65° C. for 1 minute and 72° C. for 2 minutes, and subsequent reaction at72° C. for 7 minutes were carried out using a DNA thermal cycler (PERKINELMER CETUS DNA Thermal Cycler; available from TaKaRa). As a template,20 ng of the plasmid pBS-G4 constructed in Example 3 was used.

A reaction solution was subjected to agarose gel electrophoresis torecover a DNA fragment of about 1.0 kb. A plasmid pT7B-G4sec (No. 13)was constructed by inserting the DNA fragment into a T-vector pT7Blue(manufactured by Novagen).

The absence of errors by the PCR was confirmed by determining nucleotidesequence of the DNA fragment inserted into pT7B-G4sec (No. 13).

Since the primers are designed such that a BamHI site is generated inG4-SF, and a NotI site in G4-SR, a PCR-amplified fragment moiety can becut out by digesting pT7B-G4sec (No. 13) with restriction enzymes BamHIand NotI. By digesting pT7B-G4sec (No. 13) with restriction enzymesBamHI and NotI, a BamHI-NotI fragment of 1.0 kb encoding a regionconsidered to have the catalytic activity of G4 polypeptide (from the38th glutamic acid to the 372nd tyrosine in SEQ ID NO:2) was obtained.On the other hand, a BamHI-NotI fragment of 8.9 kb was obtained bydigesting the plasmid pAMoF2-i52S with restriction enzymes BamHI andNotI. By linking these two fragments, pAMoF2-G4 was constructed (FIG.11).

(4) Secreted Production of FLAG Peptide-Fused G4 Polypeptide in NamalwaKJM-1 Cell

A control plasmid pAMoF2 and a plasmid pAMoF2-G4 for secretionexpression of FLAG peptide-fused G4 polypeptide constructed in the abovewere prepared using a plasmid preparation kit manufactured by Qiagen(/plasmid/maxi kit; trademark number 41031).

The thus prepared plasmid was subjected to ethanol precipitation andthen dissolved in TE buffer to give a concentration of 1 μg/μl.

A stable transformant was obtained by introducing each plasmid intoNamalwa KJM-1 cell by the method described in Example 7.

The thus obtained transformant was suspended in 30 ml of RPMI 1640medium containing 0.5 mg/ml G418 and 2% fetal calf serum to give adensity of 5×10⁴ cells/ml, followed by culturing at 37° C. for 10 daysin a CO₂ incubator.

After culturing, the cells were removed by centrifugation at 160×g for10 minutes and 1,500×g for 10 minutes to recover the supernatant. Theculture supernatant can be stored at −80° C. and used by thawing it,when used.

Since a β1,3-N-acetylglucosaminyltransferase encoded by the plasmidpAMoF2-G4 is expressed as a secreted form fused with the FLAG peptide,it can be easily purified using Anti-FLAG M1 Affinity Gel (manufacturedby Cosmo Bio).

To the culture supernatant obtained in the above, sodium azide, sodiumchloride and calcium chloride were added to give final concentrations of0.1%, 150 mmol/l and 2 mmol/l, respectively, and then 30 μl of Anti-FLAGM1 Affinity Gel (manufactured by Cosmo Bio) was added thereto, followedby gently stirring overnight at 4° C. After stirring, Anti-FLAG M1Affinity Gel was recovered by centrifugation at 160×g for 10 minutes,and the gel was washed twice with 1 ml of a buffer containing 50 mmol/lTris-HCl (pH 7.4), 150 mmol/l sodium chloride and 1 mmol/l calciumchloride.

After washing, the gel was treated at 4° C. for 30 minutes by adding 30μl of a buffer containing 50 mmol/l Tris-HCl (pH 7.4), 150 mmol/l sodiumchloride and 2 mmol/l EDTA to thereby elute the protein adsorbed to thegel. Thereafter, a supernatant was obtained by centrifugation at 160×gfor 10 minutes. The gel was again mixed with 30 μl of the buffercontaining 50 mmol/l of Tris-HCl (pH 7.4), 150 mmol/l of sodium chlorideand 2 mmol/l of EDTA, treated at 4° C. for 10 minutes and thencentrifuged at 160×g for 10 minutes to obtain the supernatant.Thereafter, this step was repeated again to carry out the elution stepthree times. To the eluate was added 1 mol/l calcium chloride to give afinal concentration of 4 mmol/l.

(5) Measurement of a β1,3-N-acetylglucosaminyltransferase Activity of aFLAG Peptide-Fused G4 Polypeptide

A β1,3-N-acetylglucosaminyltransferase activity of FLAG peptide-fused G4polypeptide expressed as a secreted form in Namalwa KJM-1 cell wasmeasured by using 15 μl of the eluate prepared in the above (4). Themethod of Example 8 was used in the activity measurement. As a result, aβ1,3-N-acetylglucosaminyltransferase activity was detected when theeluate derived from the culture supernatant of pAMoF2-G4-introducedNamalwa KJM-1 cell was used. The ratio of the substrate converted intothe product was 0.51%. On the other hand, the activity was not detectedwhen the eluate derived from the culture supernatant of Namalwa KJM-1cell transformed by the vector pAMoF2 was used.

Based on the above result, it was shown that the FLAG peptide-fused G4polypeptide expressed as a secreted form in Namalwa KJM-1 cell has aβ1,3-N-acetylglucosaminyltransferase activity. The result shows that aβ1,3-N-acetylglucosaminyltransferase (G4) can be produced as a secretedform fused with FLAG peptide in animal cell and that the produced fusionprotein can be easily purified using Anti-FLAG M1 Affinity Gel. It alsoshows that synthesis of sugar chains can be made by using the producedfusion protein.

Example 10 Secreted Production of a FLAG Peptide-Fusedβ1,3-N-acetylglucosaminyltransferase (G4) by Using Insect Cell as a Host

Secreted expression of the FLAG peptide-fused G4 polypeptide shown inExample 8 in an insect cell was carried out.

(1) Production of a Recombinant Virus for Secreted Expression of theFLAG Peptide-Fused G4 Polypeptide in an Insect Cell

A recombinant virus was produced by two steps comprising a step (step 1)in which a DNA encoding the protein of interest is inserted into aspecific plasmid which is called a transfer vector and a step (step 2)in which the DNA-inserted transfer vector prepared in the step 1 and awild type virus are co-transfected into an insect cell to obtain therecombinant virus by homologous recombination. The steps were carriedout by the following procedure by using BaculoGold Starter Kitmanufactured by PharMingen (product number PM-21001K).

Step 1 Insertion of a DNA Encoding a Secretable FLAG Peptide-Fused G4Polypeptide into Transfer Vector (FIG. 12).

A plasmid pVL1393-F2G4 was constructed by inserting a DNA encoding theFLAG peptide-fused secretory G4 polypeptide shown in Example 9 betweenBamHI site and NotI site of a transfer vector pVL1393 (manufactured byPharMingen).

pAMoF2-G4 prepared in Example 9 was digested with restriction enzymesHindIII and NotI to obtain a HindIII-NotI fragment of 1.05 kb.

pVL1393 origin was digested with restriction enzymes BamHI and BstPI toobtain a BamHI-BstPI fragment of

pVL1393 origin was digested with restriction enzymes NotI and BstPI toobtain a NotI-BstPI fragment of 6.4 kb.

DNAs shown in SEQ ID NOs:28 and 29 were synthesized as linkers forlinking BamHI site and HindIII site, and 5′-end phosphorylation wascarried out using T4 polynucleotide kinase.

pVL1393-F2G4 was constructed by linking these three fragments andlinkers (FIG. 12).

Step 2 Preparation of a Recombinant Virus

A recombinant baculovirus was prepared in the following manner byintroducing a filamentous baculovirus DNA (BaculoGold baculovirus DNA,manufactured by PharMihgen) and the above plasmid pVL1393-F2G4 into aninsect cell Sf9 (manufactured by PharMingen) cultured in TNM-FH insectmedium (manufactured by PharMingen), by the lipofectin method [Protein,Nucleic Acid and Enzyme, 37, 2701 (1992)].

After dissolving 1 to 5 μl of pVL1393-F2G4 and 15 ng of the filamentousbaculovirus DNA in 12 μl of distilled water, a mixture of 6 μl (6 μl) oflipofectin (manufactured by GIBCO BRL) with 6 μl of distilled water wasadded thereto and allowed to stand at room temperature for 15 minutes.

About 2×10⁶ of Sf9 cells were suspended in 2 ml of Sf900-II medium(manufactured by GIBCO BRL), and put into a cell culture plastic dishhaving a diameter of 35 mm, and an entire volume of the mixed solutionof pVL1393-F2G4, filamentous baculovirus DNA and lipofectin was addedthereto, followed by culturing at 27° C. for 3 days

One milliliter of culture supernatant containing a recombinant virus wascollected from the culture.

One milliliter of the TNM-FH insect medium was newly added to the dishafter obtaining the culture supernatant, followed by further culturingat 27° C. for 4 days. After culturing, 1.5 ml of culture supernatantcontaining the recombinant virus was collected in the same manner.

(2) Acquisition of a Recombinant Virus Solution

About 8×10⁶ of Sf9 cells were suspended in 5 ml of EX-CELL 400 medium(manufactured by JRH), put into a 25 cm² flask (manufactured by GREINER)and allowed to stand at room temperature for 30 minutes to adhere thecells to the flask, and then the supernatant was discarded and 1 ml ofEX-CELL 400 medium and 1 ml of the recombinant virus-containing culturesupernatant obtained in the above (1) were added to the resulting flask.

After the addition, the cells and virus particles were thoroughlybrought into contact by gently shaking at room temperature for 1 hourand then 4 ml of the TNM-FH insect medium was added thereto, followed byculturing at 27° C. for 4 days.

By centrifuging the culture at 1,500×g for 10 minutes, recombinantvirus-infected Sf9 cells and 5.5 ml of a solution containing recombinantvirus particles were obtained.

About 2×10⁷ of Sf9 cells were suspended in 15 ml of EX-CELL 400 medium,put into a 75 cm² flask (manufactured by GREINER) and allowed to standat room temperature for 30 minutes to adhere the cells to the flask, andthen the supernatant was discarded and 5 ml of EX-CELL 400 medium and 1ml of the recombinant virus solution obtained in the above were added tothe resulting flask.

After the addition, the cells and virus particles were thoroughlybrought into contact by gently shaking at room temperature for 1 hourand then 10 ml of the TNM-FH insect medium was added thereto, followedby culturing at 27° C. for 4 days. By centrifuging the culture at1,500×g for 10 minutes, recombinant virus-infected Sf9 cells and 15 mlof a solution containing recombinant virus particles were obtained.

The virus titer of the recombinant virus solution can be calculated bythe following method (BaculoGold Starter Kit Manual provided byPharMingen).

About 6×10⁷ of Sf9 cells are suspended in 4 ml of EX-CELL 400 medium,put into a cell culture plastic dish of 60 mm in diameter and allowed tostand at room temperature for 30 minutes to adhere the cells to thedish, and then the supernatant is discarded and 400 μl of EX-CELL 400medium and 100 μl of the recombinant virus solution diluted to 10⁻⁴ or10⁻⁵ with EX-CELL 400 medium are added to the resulting dish.

After the addition, the cells and virus particles are thoroughly broughtinto contact by gently shaking the dish at room temperature for 1 hour.

After the contact, the medium is removed from the dish, and a mixedsolution of 2 ml of EX-CELL 400 medium containing 2% low melting pointagarose (Agarplaque Agarose, manufactured by PharMingen) (kept at 42°C.) with 2 ml of TNM-FH Insect Medium (kept at 42° C.) is poured intothe dish and allowed to stand at room temperature for 15 minutes.

After standing, the dish is wrapped with, a vinyl tape in order toprevent drying, and the resulting dish is put into a sealable plasticcontainer, followed by culturing at 27° C. for 5 days.

After culturing, 1 ml of PBS buffer containing 0.01% Neutral Red isadded to the dish, followed by, culturing for 1 day, and then the numberof formed plaques is counted.

(3) Secreted Production and Purification of a FLAG Peptide-Fused G4Polypeptide

Since the G4 polypeptide encoded by the recombinant virus derived from aplasmid pVL1393-F-2G4 is expressed as a secreted form fused with FLAGpeptide, it can be easily purified using Anti-FLAG M1 Affinity Gel(manufactured by Cosmo Bio).

About 2×10⁷ of Sf21 cells were suspended in 15 ml of EX-CELL 400 medium,put into a 75 cm² flask (manufactured by GREINER) and allowed to standat room temperature for 30 minutes to adhere the cells to the flask, andthen the supernatant was discarded and 4 ml of EX-CELL 400 medium and 1ml of the recombinant virus solution obtained in the above (2) wereadded to the resulting flask.

After the addition, the cells and virus particles were thoroughlybrought into contact by gently shaking at room temperature for 1 hour,and then 10 ml of the TNM-FH insect medium was added thereto, followedby culturing at 27° C. for 4 days. By centrifuging the culture at1,500×g for 10 minutes, 15 ml of a culture supernatant possiblycontaining the secreted G4 was obtained.

To 30 ml of the culture supernatant obtained in the above, sodium azide,sodium chloride and calcium chloride were added to give finalconcentrations of 0.1%, 150 mmol/l and 2 mmol/l, respectively, and thenthe mixture was mixed with 30 μl of Anti-FLAG M1 Affinity Gel(manufactured by Cosmo Bio), followed by gently stirring overnight at 4°C.

After stirring, Anti-FLAG M1 Affinity Gel was recovered by 10 minutes ofcentrifugation at 160×g, and the gel was washed twice with 1 ml of abuffer containing 50 mmol/l of Tris-HCl (pH 7.4), 150 mmol/l of sodiumchloride and 1 mmol/l of calcium chloride.

After the washing, the gel was treated at 4° C. for 30 minutes by adding80 μl of a buffer containing 50 mmol/l Tris-HCl (pH 7.4), 150 mmol/lsodium chloride and 2 mmol/l EDTA to thereby elute the protein adsorbedto the gel. Thereafter, a supernatant was obtained by centrifugation at160×g for 10 minutes. The gel was again mixed with 80 μl of the buffercontaining 50 mmol/l Tris-HCl (pH 7.4), 150 mmol/l sodium chloride and 2mmol/l EDTA, treated at 4° C. for 10 minutes, followed by centrifugingat 160×g for 10 minutes to obtain the supernatant. Thereafter, this,step was repeated again to carry out the elution step three times. Tothe eluate, 1 mol/l calcium chloride was added to give a finalconcentration of 4 mmol/l.

SDS-PAGE was carried out by using 15 μl of the thus prepared eluate, andthen staining was carried out by using Coomassie Brilliant Blue (FIG.13).

When the eluate prepared from the culture supernatant of Sf21 infectedwith the recombinant virus derived from pVL1393-F2G4 was used, a broadband ranging from 43 to 48 kD was found. On the other hand, such a bandwas not detected when the eluate prepared from the culture supernatantof Sf21 infected with the recombinant virus derived from a vectorpVL1393 was used.

Based on the above result, it was shown that the FLAG peptide-fused G4polypeptide is produced as a secreted form in culture supernatant andcan be purified easily using Anti-FLAG M1 Affinity Gel.

(4) Measurement of β1,3-N-acetylglucosaminyltransferase Activity of theFLAG Peptide-Fused G4 Polypeptide

A β1,3-N-acetylglucosaminyltransferase activity of FLAG peptide-fused G4polypeptide produced as a secreted form in the insect cell was measuredusing 15 μl of the eluate prepared in the above (3). The method ofExample 8 was used for the activity measurement. As a result, aβ1,3-N-acetylglucosaminyltransferase activity was detected when theeluate derived from the culture supernatant of the insect celltransformed by pVL1393-F2G4 was used. The ratio of the substrateconverted into the product was 12.1%.

A β1,3-N-acetylglucosaminyltransferase activity was also detected whenthe resin before elution was used. The ratio of the substrate convertedinto the product was 21.0%. The result shows that a sugar chain can besynthesized even under conditions in which the enzyme is adsorbed to theresin.

On the other hand, the activity was not detected when the eluate derivedfrom the culture supernatant of the insect cell introduced with a vectorpVL1393 was used.

Based on the above result, it was shown that the FLAG peptide-fused G4polypeptide expressed as a secreted form in the insect cell has aβ1,3-N-acetylglucosaminyltransferase activity. The result shows that aβ1,3-N-acetylglucosaminyltransferase (G4) can be produced as a secretedform fused with FLAG peptide in insect and that the produced fusionprotein can be easily purified using Anti-FLAG M1 Affinity Gel. It alsoshows that synthesis of sugar chains can be made by using the producedfusion protein.

It was found from these results that the productivity is high whenproduced in the insect cell in comparison with the case of producing inNamalwa KJM-1 cell.

Example 11 Examination on the Substrate Specificity of a Secretoryβ1,3-N-acetylglucosaminyltransferase (Secretory G4)

Examination on the substrate specificity of aβ1,3-N-acetylglucosaminyltransferase (G4) was carried out by using theFLAG peptide-fused G4 polypeptide produced as a secreted form in Example10.

(1) Analysis Using Pyridylaminated Oligosaccharides as Substrates

The method shown in Example 8 was used as the activity measuring method.Specifically, after the reaction at 37° C. for 14.5 hours in 30 μl of anassay solution [200 mmol/l of MOPS (pH 7.5), 20 mmol/l of UDP-GlcNAc(SIGMA), 20 mmol/l of MnCl₂, 50 μmol/l of pyridylaminated sugar chainsubstrate and 15 μl of the above eluate], products were detected byHPLC. As substrates, LNnT, lacto-N-tetraose(Galβ1-3GlcNAcβ1-3Galβ1-4Glc, hereinafter referred to as “LNT”),lacto-N-fucopentaose II (Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc,hereinafter referred to as “LNFP-II”), lacto-N-fucopentaose III(Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc, hereinafter referred to as“LNFP-III”), lacto-N-fucopentaose V(Galβ1-3GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc, hereinafter referred to as“LNFP-V”) and lacto-N-difucohexaose II(Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc, hereinafter referred toas “LNDFH-II”) (all manufactured by oxford Glycosystems) were used afterfluorescently labeling them with aminopyridine. Fluorescence labeling ofthese substrates were carried out in accordance with the usual method[Agric. Biol. Chem., 54, 2169 (1990)].

For each substrate, the reaction was carried using the assay solutionwith or without UDP-GlcNAc (sugar donor), and then HPLC was carried outand peaks appeared only in the assay solution containing UDP-GlcNAc weredefined as products.

The assay solution after completion of the reaction was treated at 100°C. for 5 minutes and then centrifuged at 10,000×g for 5 minutes, and aportion (5 μl) of the thus obtained supernatant was applied to HPLC.

The HPLC was carried out by using TSK-gel ODS-80Ts column (4.6×300 mm;Tosoh); 0.02 mol/l ammonium acetate buffer (pH 4.0) was used as theeluate at an elution temperature of 50° C. and at a flow rate of 0.5ml/min.

Products were detected by using a fluorescence spectrum photometerFP-920 (JASCO Corporation) (excitation wavelength 320 nm, radiationwavelength 400 nm).

Relative activities when the activity on LNnT used as a substrate is,defined as 100% are shown in Table 1. When LNnT was used as a substrate,the conversion ratio of the substrate into the product was 12.9%. It wasfound that the β1,3-N-acetylglucosaminyltransferase (G4) uses LNT andLNFP-V as a good substrate in addition to LNnT. It is known that alreadyknown β1,3-N-acetylglucosaminyltransferases use LNnT as a good substratebut hardly use LNT as a substrate [J. Biol. Chem., 268, 27118 (1993),Proc. Natl. Acad. Sci. USA, 96, 406 (1999)]. In consequence, it wasfound that a β1,3-N-acetylglucosaminyltransferase (G4) is an enzymehaving a substrate specificity which is clearly different from that ofthe already known β1,3-N-acetylglucosaminyltransferases.

It is known that a cancer-related sugar chain having dimeric Lewis asugar chain antigen [Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAc]is expressed in large bowel cancer tissues and large bowel cancer celllines, for example, the presence of a glycolipid having a structure ofGalβ3(Fucα1-4)GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-3Glc-Cer hasbeen found [J. Biol. Chem., 266, 8439-8446 (1991)]. Since theβ1,-3N-acetylglucosaminyltransferase (G4) can efficiently transferN-acetylglucosamine to the terminal galactose residue of theGalβ1-3GlcNAc structure too, it is considered that aβ1,3-N-acetylglucosaminyltransferase (G4), but not the already knownβ1,3-N-acetylglucosaminyltransferases involved in the synthesis of thecore sugar chain of the dimeric Lewis a sugar chain. As will bedescribed later in Example 13(3) in detail, the G4 transcriptionproducts are highly expressed in a large bowel cancer cell line. TABLE 1Substrate specificity of a β1,3-N-acetylglucosaminyltransferase (G4)with pyridylaminated oligosaccharides as substrates Relative SubstrateSugar chain structure activity (%) LNnT Galβ1-4GlcNAcβ1-3Galβ1-4Glc 100LNFP-III Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc 4.7 LNTGalβ1-3GlcNAcβ1-3Galβ1-4Glc 84.5 LNFP-IIGalβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc 0 LNFP-VGalβ1-3GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc 65.1 LNDFH-IIGalβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1- 0 4(Fucα1-3)Glc(2) Analysis Using Unlabeled Oligosaccharides as Substrates

The reaction using the glycosyltransferase was carried out as follows.The reaction was carried out at 37° C. for 16 hours in 40 μl of an assaysolution [50 mmol/l MOPS (pH 7.5), 5 mmol/l UDP-GlcNAc (SIGMA), 5 mmol/lMnCl₂, 10 mmol/l sugar chain substrate and 10 μl of the above eluate].Next, the reaction solution was treated at 100° C. for 5 minutes andthen centrifuged at 10,000×g for 20 minutes to obtain the supernatant,and a portion thereof was analyzed using HPAE/PAD (High PerformanceAnion Pulsed Amperometoric Detection; manufactured by DIONEX). Thespecific method was carried out in the usual way [Anal. Biochem., 189,151 (1990), J. Biol. Chem., 273, 433 (1998)].

As substrates, the following unlabeled oligosaccharides were used:lactose (Galβ1-4Glc), N-acetyllactosamine (Galβ1-4GlcNAc, hereinafterreferred to as “LacNAc” in some cases), LNnT, LNT and lacto-N-neohexaose(Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc, hereinafter referred toas “LNnH”).

For each substrate, the reaction was carried using the assay solutionwith or without UDP-GlcNAc (sugar donor), and then HPLC was carried outand peaks appeared only in the assay solution containing UDP-GlcNAc weredefined as products.

In identifying the product, coincidence of its elution time with that ofa standard sugar chain was used as the index. As the standard sugarchains, GlcNAcβ1-3Galβ1-4Glc, GlcNAcβ1-3Galβ1-4GlcNAc andGlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc were used.

Relative activities when the activity on LNnT used as a substrate isdefined as 100% are shown in Table 2. When LNnT was used as a substrate,the conversion ratio of the substrate into the product was 1.7%. It wasfound that the β1,3-N-acetylglucosaminyltransferase (G4) uses LNT, LNnHand Galβ1-4Glc as good substrates in addition to LNnT. On the otherhand, it did not use Galβ1-4GlcNAc as a substrate. It is known thatalready known β1,3-N-acetylglucosaminyltransferases use both Galβ1-4Glcand Galβ1-4GlcNAc as good substrates [J. Biol. Chem., 268, 27118 (1993),Proc. Natl. Acad. Sci. USA, 96, 406 (1999)]. In consequence, it wasfound that a β1,3-N-acetylglucosaminyltransferase (G4) is an enzymehaving a substrate specificity which is clearly different from that ofthe already known enzymes. TABLE 2 Substrate specificity of aβ1,3-N-acetylglucosaminyltransferase (G4) with unlabeledoligosaccharides as substrates Relative Substrate Sugar chain structureactivity (%) LNnT Galβ1-4GlcNAcβ1-3Galβ1-4Glc 100 LNTGalβ1-3GlcNAcβ1-3Galβ1-4Glc 114 LNnH Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-45 3Galβ1-4Glc Lactose Galβ1-4Glc 235 LacNAc Galβ1-4GlcNAc 0

On the other hand, when β1,3-galactosyltransferase activity of thesecretory G4 was measured by using GlcNAc and GlcNAcβ1-3Galβ1-4Glc asreceptor substrates, the activity was not detected.

The glycosyltransferase reaction was carried out as follows. Thereaction was carried out at 37° C. for 16 hours in 40 μl of an assaysolution [50 mmol/l MOPS (pH 7.5), 5 mmol/l UDP-GlcNAc (SIGMA), 5 mmol/lMnCl₂, 10 mmol/l of a sugar chain substrate and 10 μl of the aboveeluate]. Next, the reaction solution was treated at 100° C. for 5minutes and then centrifuged at 10,000×g for 20 minutes to obtain thesupernatant, and a portion thereof was analyzed by using HPAE/PAD (HighPerformance Anion Pulsed Amperometoric Detection; manufactured byDIONEX). The specific method was carried out in the usual way [Anal.Biochem., 189, 151 (1990), J. Biol. Chem., 273, 433 (1998)].

Example 12 Synthesis of a poly-N-acetyllactosamine Sugar Chain UsingSecretory β1,3-N-acetylglucosaminyltransferase (Secretory G4)

Synthesis of poly-N-acetyllactosamine sugar chain was carried out byusing the FLAG peptide-fused G4 polypeptide produced as a secreted formin Example 10 and β1,4-galactosyltransferase.

(1) Two Step Reaction

By allowing the FLAG peptide-fused G4 polypeptide produced as a secretedform in Example 10 to react with LNnT, a sugar chain in whichN-acetylglucosamine was added via a β1,3-linkage to the non-reducingterminal galactose residue of LNnT(GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc) was synthesized. Next, byallowing β1,4-galactosyltransferase (manufactured by SIGMA) purifiedfrom bovine milk to react with the sugar chain, a sugar chain in whichgalactose was added via a β1,4-linkage to the non-reducing terminalN-acetylglucosamine residue(Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc) was synthesized. In thesame manner, Galβ1-4GlcNAcβ1-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc wassynthesized by using LNT as a substrate. Also,Galβ1-4GlcNAcβ1-3Galβ1-3GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc was synthesized byusing LNFP-V [(Galβ1-3GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc) as a substrate. Thespecific reaction was carried out as follows.

The reaction was carried out at 37° C. for 14.5 hours in 30 μl of areaction solution [200 mmol/l MOPS (pH 7.5), 20 mmol/l UDP-GlcNAc(SIGMA), 20 mmol/l MnCl₂, 50 μmol/l of a pyridylaminated sugar chainsubstrate and 15 μl of the above eluate]. As a substrate, LNnT, LNT orLNFP-V was used. After confirming formation of products by HPLC in thesame manner as the method described in Example 11,β1,4-galactosyltransferase (20 mU) and UDP-Gal (20 mmol/l) were added tothe reaction solution and allowed to react at 37° C. for 14.5 hours.Formation of products was confirmed by HPLC.

(2) One-Pot Reaction

A sugar chain in which N-acetylglucosamine was added to the non-reducingend of the sugar chain (Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc)was synthesized by allowing the FLAG peptide-fused G4 polypeptideproduced as a secreted form in Example 10 to react simultaneously withLNnT and β1,4-galactosyltransferase (manufactured by SIGMA) purifiedfrom bovine milk. In the same manner,Galβ1-4GlcNAcβ1-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc was synthesized using LNTas a substrate. Also,Galβ1-4GlcNAcβ1-3Galβ1-3GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc was synthesized byusing LNFP-V [(Galβ1-3GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc) as a substrate. Thespecific reaction was carried out as follows.

The reaction was carried out at 37° C. for 14.5 hours in 30 μl of areaction solution [200 mmol/l MOPS (pH 7.5), 20 mmol/l UDP-GlcNAc(SIGMA), 20 mmol/l UDP-Gal (SIGMA), 20 mmol/l MnCl₂, 50 μmol/l of apyridylaminated sugar chain substrate, 10 μl of the above eluate and 20mU β1,4-galactosyltransferase]. As a substrate, LNnT, LNT or LNFP-V wasused. Formation of products were confirmed by HPLC.

Example 13 Examination of the Expression Level of Transcripts of EachGene for G3, G4 or G7 in Various Cells

The transcription products of each gene for G3, G4 (G4-2) or G7 weredetermined by a semi-quantitative PCR method in accordance with a usualmethod [PCR Protocols, Academic, Press (1990)]. Also, transcriptionproducts of β-actin considered to be expressed in each cell at a similardegree was simultaneously mesured to thereby confirm that there are nogreat differences in the amount of mRNA among cells and the conversionefficiency of single-stranded cDNA from mRNA by reverse transcriptaseamong samples.

Transcription products of β-actin were mesured by a quantitative PCRmethod in the usual way [Proc. Natl. Acad. Sci. USA, 87, 2725 (1990), J.Biol. Chem., 269, 14730 (1994), Japanese Published Unexamined PatentApplication No. 181759/94].

(1) Synthesis of Single-Stranded cDNAs Derived from Various Cells andCell Lines

As cell lines, colon cancer cell lines (WiDR, Colo205, SW1116, LS180,DLD-1), lung cancer cell lines (QG90, HLC-1, PC9), pancreatic cancercell lines (Capan-1, Capan-2), a prostate cancer cell line PC-3, agastric cancer cell line KATO III, T-cell lines (Jurkat, CCRF-CEM,HSB-2, PEER, Molt-3, Molt-4, HUT78, HPB-ALL), B-cell lines (NamalwaKJM-1, Daudi, Wa, CCRF-SB, Jiyoye, RPMI1788, RPMI8226, HO328-8, BALL-1,KOPN-K, IM-9), a melanoma cell line WM266-4, granulocyte/monocyte celllines (THP-1, HL-60, U-937) and a neuroblastoma cell line SK-N-MC wereused. QG90, HPB-ALL, Wa, SW1116 and Jurkat were obtained from AichiCancer. Center. HLC-1 was obtained from Cancer Research Institute, OsakaUniversity. HO328-8 was obtained from Food Chemistry, Department ofAgriculture, Kyushu University. KOPN-K was obtained from Saitama CentralHospital. KATO III and PC-9 were obtained from Immune Biology ResearchInstitute. CCRF-SB and RPMI8226 were obtained from DainipponPharmaceutical. BALL-1, PEER, Molt-4, Daudi, IM-9 and KY821 wereobtained from JCRB. Other cells were obtained from ATCC.

Jurkat cells stimulated with phytohemagglutinin-P (PHA-P) and12-O-tetradecanoylphorbol 13-acetate (TPA) were prepared as follows.Jurkat cells inoculated in a density of 4×10⁵ cells/ml into RPMI 1640medium containing 10% FCS were added with 1 μg/ml of PHA-P and 50 ng/mlof TPA, followed by culturing for 3 hours, 12 hours or 24 hours, andthen the cells were recovered.

Also, polymorphonuclear leukocytes and mononuclear leukocytes wereobtained by separating them from peripheral blood of a healthy adult byuse of Polymorphprep™ as a kit manufactured by Nycomed Pharma. The thusobtained mononuclear leukocytes were further separated into monocyte andlymphocyte in accordance with the general method [J. Immunol., 130, 706(1983)].

Total RNA of each cell was prepared in accordance with the generalmethod [Biochemistry., 18, 5294 (1977)]. Single-stranded cDNA, wassynthesized from the total RNA using a kit (SUPER™ PreamplificationSystem; manufactured by BRL). Single-stranded cDNA samples weresynthesized from 5 μg of total RNA regarding the cell lines, and from 1μg of total RNA regarding the leukocytes, and diluted 50 times and 10times, respectively with water to be used as templates of PCR. Asprimers, an oligo(dT) primer or random primers were used. RegardingSK-N-MC, SK-N-SH, Colo205, SW1116, LS180, DLD-1, Capan-1 and Capan-2,random primers were used. An Oligo(dT) primer was used in other cases,

Also, single-stranded cDNAs were synthesized from mRNA preparations(manufactured by Clontech) derived from various human organs.Single-stranded cDNAs were synthesized from 1 μl of mRNA, diluted 240times with water and used as a template of PCR. As a primers, anoligo(dT) primers was used. As mRNAs, mRNAs derived from the following35 organs were used; 1: adrenal gland; 2: brain; 3: caudate nucleus; 4:hippocampus; 5: substantia nigra; 6: thalamus; 7: kidney; 8: pancreas;9: pituitary gland; 10: small intestine; 11: bone marrow; 12: amygdala;13: cerebellum; 14: callosal body; 15: fetal brain; 16: fetal kidney;17: fetal liver; 18: fetal lung; 19: heart; 20: liver; 21: lung; 22:lymph node; 23: mammary gland; 24: placenta; 25: prostate; 26: salivarygland; 27: skeletal muscle; 28: spinal cord; 29: spleen; 30: stomach;31: testis; 32: thymus; 33: thyroi; 34: trachea; and 35: uterus.

(2) Preparation of Standard and Internal Control for Quantitative PCR

Each of pBS-G3, pBS-G4-2 and pT7B-G7 was converted into linear DNA bydigesting it with restriction enzymes capable of cutting out the cDNAmoiety and then used as the standard for the determination. Afterconfirming complete cutting of each plasmid, it was used by diluting itstepwise with water containing 1 μl/ml yeast transfer RNA. Specifically,pBS-G3 was digested with NotI and SalI, pBS-G4-2 with NotI and pT7B-G7with HincII and SmaI.

Also, each of pUC119-ACT and pUC119-ACTd was converted into linear DNAby digesting it with restriction enzymes (HindIII and Asp718) capable ofcutting out the cDNA moiety and then used respectively as the standardand internal control for the determination of transcription product ofβ-actin [J. Biol. Chem., 269, 14730 (1994), Japanese PublishedUnexamined Patent Application No. 181759/94]. After confirming completecutting of each plasmid, it was used by diluting it stepwise with watercontaining 1 μl/ml yeast transfer RNA.

(3) Determination of the Amount of Transcripts of Each Gene for G3, G4or G7 by PCR

PCR was carried out by using each single-stranded cDNAs prepared in theabove (1) from various cells and cell lines as a template. As primersfor PCR, F-3-5 and R-3-5 were used for detecting G3 transcriptionproduct, and F-4-5 and R-4-5 for detecting G4 transcription product andF-7-3a (SEQ ID NO:30) and R-7-3a for detecting G7 transcript. Also, acalibration curve was prepared by carrying out PCR in the same manner byusing the standard prepared in the above (2) as templates.

The PCR was carried out by using Recombinant Taq DNA Polymerase (GeneTaq) manufactured by Nippon Gene, and 10× Gene Taq Universal Buffer and2.5 mmol/l dNTP Mixture attached thereto, in accordance with theinstructions. The reaction was carried out by using 20 μl of a reactionsolution to which dimethyl sulfoxide was added to give a finalconcentration of 5%.

The reaction solution (19 μl) containing respective components otherthan Taq DNA polymerase was treated at, 97° C. for 3 minutes by using athermal cycler (DNA Enzine PTC-200 Peltier Thermal Cycler) manufacturedby MJ RESEARCH and then rapidly cooled in ice. Next, 1 μl of 1/5-dilutedTaq DNA polymerase was added to the reaction solution and then thereaction of 94° C. for 30 seconds, 60° C. for 1 minute and 72° C. for 2minutes was repeated 26 to 30 cycles by using the thermal cycler of MJRESEARCH. A portion (7 μl) of the reaction solution was subjected toagarose gel electrophoresis and then the gel was stained with SYBR GreenI nucleic acid stain (manufactured by Molecular Probes). The amounts ofthe amplified DNA fragments were measured by analyzing patterns of theamplified DNA fragments by a fluoro imager (FluorImager SI; manufacturedby Molecular Dynamics). In order to determine the amount of transcriptsmore accurately, similar PCR was carried out by changing the number ofcycles of PCR. The amount of the standard was changed depending on thenumber of cycles of PCR.

The amount of β-actin transcript was determined in the same manner asdescribed in literatures [J. Biol. Chem., 269, 14739 (1994), JapanesePublished Unexamined Patent Application No. 181759/94].

Although the expression level was low, G3 transcripts was expressed inall of the 35 human organs examined (FIG. 14). Its expression was alsofound in various human leukocyte cell lines and the polymorphonuclearleukocytes, monocytes and lymphocytes prepared from human peripheralblood (FIG. 15).

G4 transcript was expressed in a small amount in trachea, placenta andstomach among the 35 human organs shown in the above (1) (FIG. 16). Itsexpression was not found in various human leukocyte cell lines andpolymorphonuclear leukocytes and lymphocytes prepared from humanperipheral blood (FIG. 17).

On the other hand, a relatively large amount of expression was found incolon cancer cell lines (WiDR, Colo205, SW1116, LS180, DLD-1), lungcancer cell lines (QG90, HLC-1, PC9), pancreatic cancer cell lines(Capan-1, Capan-2) and a gastric cancer cell line KATO III (FIG. 18). Asan example, results of the determination are shown below. Valuesexpressed as the ratio (%) of the expression level of G4 transcript inWiDR, QG90, PC-3, HLC-1, PC9, KATO III, SK-N-MC, SK-N-SH, Colo205,SW1116, LS180, DLD-1, Capan-1 and Capan-2 based on the expressed amountof β-actin transcript are 0.49, 0.29, 0.069, 0.34, 0.35, 0.94, 0.027,0.0037, 0.10, 4.6, 0.95, 0.85, 0.67 and 0.92 in this order.

G7 transcript was expressed in a small amount in brain, caudate nucleus,hippocampus, kidney, amygdala, cerebellum and fetal brain among the 35human organs shown in the above (1) (FIG. 19). Its expression was hardlyfound in polymorphonuclear leukocytes, monocytes and lymphocytesprepared from human peripheral blood (FIG. 20). Among the T-cell lines,a small amount of expression was found only in Jurkat and HPB-ALL, butthe expression was not found in the B-cell lines (FIG. 20).

On the other hand, a relatively large amount of expression was found ina neuroblastoma cell line SK-N-MC and a prostate cancer cell line PC-3.A small amount of expression was found in colon cancer cell lines(Colo205, SW1116 and LS180) and pancreatic cancer cell lines (Capan-1and Capan-2).

Based on the results, it was found that expression pattern of respectivegenes for G3, G4 and G7 is different from one another. Thus, it isconsidered that each of these three genes for G3, G4 and G7 encode aβ1,3-N-acetylglucosaminyltransferase but each gene takes part indifferent functions in different tissues.

Since the expression level of the G3 transcript was large in leukocytes,it was suggested that G3 involved in the synthesis of apoly-N-acetyllactosamine sugar chain in leukocytes. Accordingly, thereis a possibility that inflammation can be diagnosed by examining theexpression level of G3 transcript or G3 polypeptide. Also, there is apossibility that inflammation can be controlled by controlling theexpression of the G3 gene and inhibiting the activity of G3 polypeptide.

Also, since G3 transcript is expressed in mammary gland, there is apossibility that G3 involved in the synthesis of oligosaccharideshaving, GlcNAcβ-1-3Gal structure contained in human milk such as LNnT,LNT and the like.

Regarding G4 transcript, since its expression level is increased in celllines derived from colon cancer, pancreatic cancer and gastric cancer,there is a possibility that cancers can be diagnosed by examining theexpression level of G4 transcript or G4 polypeptide. Also, there is apossibility that metastasis of cancer cells can be controlled bycontrolling the expression of G4 gene and inhibiting the activity of G4polypeptide.

Regarding G7 transcript, since its expression level is increased in celllines derived from neuroblastoma, prostatic cancer, colon cancer andpancreatic cancer, there is a possibility that diagnosis of cancers canbe made by examining the expresssion level of G7 transcript or G7polypeptide. Also, there is a possibility that metastasis of cancercells can be controlled by controlling the expression of G7 gene andinhibiting the activity of G7 polypeptide.

Also, since the expression pattern of twoβ1,3-N-acetylglucosaminyltransferases so far cloned in various cells andtissues is different from that of three novelβ1,3-N-acetylglucosaminyltransferases (G3, G4 and G7) obtained accordingto the present invention, it is considered that these three novelβ1,3-N-acetylglucosaminyltransferases have functions different fromthose of the known enzymes.

Example 14 Production of a FLAG Peptide-Fusedβ1,3-N-acetylglucosaminyltransferase (G3) as a Secreted Form UsingInsect Cell as a Host

Secreted expression of a FLAG peptide-fused G3 polypeptide in an insectcell was carried out in the same manner as in Example 10.

(1) Construction of a Plasmid pVL1393-F2G3

It was considered based on its primary sequence that G3 polypeptidecomprises an N-terminal cytoplasmic region containing 9 amino acids, asubsequent membrane-binding region rich in hydrophobic nature containing19 amino acids, a stem region containing at least 12 amino acids and theremaining C-terminal part comprising most of, the polypeptide andcontaining a catalytic region. Accordingly, secreted expression of G3polypeptide was attempted by removing the N-terminal cytoplasmic regioncontaining 9 amino acids, the membrane-binding region containing 19amino acids and a part of the stem region (2 amino acids), and adding aimmunoglobulin signal sequence and FLAG peptide to the removed regions.

First, a plasmid pBlunt-G3 was constructed by preparing a DNA regionencoding a region considered to have the catalytic activity of G3polypeptide (from the 31st serine to the 397th cysteine in SEQ ID NO:1)by PCR and then inserting it into pCR-Blunt vector (manufactured byInvitrogen). The specific method is described below.

As primers for PCR, a DNA fragment shown by SEQ ID NO:31 (hereinafterreferred to as “G3-1”) and a DNA fragment shown by SEQ IDNO:32(hereinafter referred to as “G3-2”) were synthesized. They aredesigned such that a BamHI site is generated in G3-1, and a NotI site inG3-2.

PCR was carried out by using Pyrobest DNA Polymerase manufactured byTaKaRa and 10× Pyrobest Buffer and 2.5 mmol/l dNTP Mixture attached tothe kit, in accordance with the instructions. Using a DNA thermal cycler(PERKIN ELMER CETUS DNA Thermal Cycler; manufactured by TaKaRa), 16cycles of the reaction at 94° C. for 30 seconds, 65° C. for 1 minute and72° C. for 2 minutes were carried out, followed by a reaction at 72° C.for 10 minutes. As a template, 20 ng of a plasmid pBS-G3 constructed inExample 2 was used. A DNA fragment of about 1.1 kb was obtained by thePCR. A plasmid pBlunt-G3 was constructed by inserting this DNA fragmentinto pCR-Blunt vector. The absence of errors by the PCR was confirmed bydetermining the nucleotide sequence of the DNA fragment inserted intopBlunt-G3.

By digesting pBlunt-G3 with restriction enzymes BamHI and NotI, aBamHI-NotI fragment of 1.1 kb encoding a region considered to have thecatalytic activity of G3 polypeptide (from the 31st serine to the 397thcysteine in SEQ ID NO:1) was obtained. By digesting pVL1393 withrestriction enzymes NotI and BstPI, a NotI-BstPI fragment of 6.4 kb wasobtained. By digesting a plasmid pVL1393-F2G4 constructed in Example 10with restriction enzymes BamHI and BstPI, a BamHI-BstPI fragment of 3.3kb was obtained pVL1393-F2G3 was constructed by linking these threefragments.

(2) Preparation of a Recombinant Virus

A recombinant virus was prepared for the secreted expression of the FLAGpeptide-fused G3 polypeptide in an insect cell. A recombinantbaculovirus was prepared by introducing a filamentous baculovirus DNAand the plasmid pVL1393-F2G3 constructed in the above (1) into an insectcell Sf9 by a lipofectin method. The method described in Example 10 wasused.

(3) Secreted Production and Purification of a FLAG Peptide-Fused G3Polypeptide

A FLAG peptide-fused G3 polypeptide was produced as a secreted form inthe insect cell by using the recombinant virus prepared in the above(2). Thereafter, the polypeptide was purified from a culture supernatantcontaining the polypeptide. The method described in Example 10 was used.

SDS-PAGE was carried out by using 15 μl of a purified sample, and thenstaining was carried by using Coomassie Brilliant Blue (FIG. 21). When apurified sample prepared from the culture supernatant of Sf21 infectedwith the recombinant virus derived from pVL1393-F2G3 was used, bandsranging from 51 to 56 kD were found. It is considered that difference inthe molecular weight of each band is based on the number and size of theadded sugar chain. In G3 polypeptide, there are five N-glycosylation. Onthe other hand, such bands were not detected when a sample purified fromthe culture supernatant of Sf21 infected with the recombinant virusderived from the vector pVL1393 was used in the same manner.

Based on the above result, it was shown that the FLAG peptide-fused G3polypeptide is produced as a secreted form in the insect cell culturesupernatant and can be purified easily by use of Anti-FLAG M1 AffinityGel.

(4) Measurement of a β1,3-N-acetylglucosaminyltransferase Activity of aFLAG Peptide-Fused G3 Polypeptide

A β1,3-N-acetylglucosaminyltransferase activity of FLAG peptide-fused G3polypeptide was measured by using 15 μl of the purified sample preparedin the above (3). The method of Example 8 was used for the activitymeasurement. As a result, a β1,3-N-acetylglucosaminyltransferaseactivity was detected. The ratio of the substrate converted into theproduct was 100%. On the other hand, when the sample purified from theculture supernatant of Sf21 infected with a vector pVL1393 was used inthe same manner, the activity was not detected.

Based on the above result, it was shown that the FLAG peptide-fused G3polypeptide secreted expression in the insect cell has aβ1,3-N-acetylglucosaminyltransferase activity. The result shows that aβ1,3-N-acetylglucosaminyltransferase (G3) can be produced as a secretedform fused with FLAG peptide in insect, and that synthesis of sugarchains can be made by using the produced fusion protein.

Example 15 Examination on Substrate Specificity of a Secretedβ1,3-N-acetylglucosaminyltransferase (Secretory G3)

Substrate specificity of a β1,3-N-acetylglucosaminyltransferase (G3) wasexamined using the FLAG peptide-fused G3 polypeptide purified in Example14.

(1) Analysis by Using Pyridylaminated Oligosaccharides as Substrates

Substrate specificity of the FLAG peptide-fused G3 polypeptide wasexamined by the method shown in Example 11(1). The reaction was carriedout at 37° C. for 2 hours.

Relative activities when the activity on LNnT used as a substrate isdefined as 100% are shown in Table 3. When LNnT was used as a substrate,the conversion efficiency of the substrate into the product was 82.5%.It was found that a β1,3-N-acetylglucosaminyltransferase (G3) uses LNnTas a good substrate but hardly uses LNT as a substrate. On the otherhand, it was found that an oligosaccharide LNFP-V in which fucose isadded to glucose residue in LNT via an α1,3-linkage, becomes arelatively good substrate of G3. It was found also that anoligosaccharide LNFP-III in which fucose is added via an α1,3-linkage tothe GlcNAc residue existing in the second position from the non-reducingterminal of LNnT does not become a substrate of G3. Also, it was foundthat oligosaccharides LNFP-II and LNDFH-II in which fucose is added viaan α1,4-linkage to GlcNAc residue existing in the second position fromthe non-reducing terminal of LNnT do not become substrates of G3.

By comparing Tables 1 and 3 with Table 5 which will be described later,it was also found that a β1,3-N-acetylglucosaminyltransferase (G3) is anenzyme having a substrate specificity which is clearly different fromthat of the other β1,3-N-acetylglucosaminyltransferases (G4 and G7)obtained according to the present invention. TABLE 3 Substratespecificity of a β1,3-N-acetylglucosaminyltransferase (G3) withpyridylaminated oligosaccharides as substrates Relative Substrate Sugarchain structure activity (%) LNnT Galβ1-4GlcNAcβ1-3Galβ1-4Glc 100LNFP-III Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc 0 LNTGalβ1-3GlcNAcβ1-3Galβ1-4Glc 3.7 LNFP-IIGalβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc 0 LNFP-VGalβ1-3GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc 30.8 LNDFH-IIGalβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1- 0 4(Fucα1-3)Glc(2) Analysis Using Unlabeled Oligosaccharides as Substrates

Substrate specificity of the FLAG peptide-fused G3 polypeptide wasexamined by the method shown in Example 11(2). The enzyme reaction wascarried out at 37° C. for 2 hours.

Relative activities when the activity for LNnT used as a substrate isdefined as 100% are shown in Table 4. When LNnT was used as a substrate,the conversion ratio of a substrate into the product was 4.6%. It wasfound that the β1,3-N-acetylglucosaminyltransferase (G3) uses adisaccharide lactose and a hexaose LNnH as good substrates in additionto a tetraose LNnT. Based on the above, it is considered that G3 cansynthesize a poly-N-acetyllactosamine sugar chain efficiently. Althoughthe activity was lower than the activity for LNnT, lactose and LNnH,LacNAc and LNT were also usable as substrates for G3. It is known thatalready known β1,3-N-acetylglucosaminyltransferases prefer LacNAc as agood substrate rather than lactose [J. Biol. Chem., 268, 27118 (1993),Proc. Natl. Acad. Sci. USA, 96, 406 (1999)]. In consequence, it wasfound that the β1,3-N-acetylglucosaminyltransferase (G3) is an enzymehaving a substrate specificity which is clearly different from that ofthe already known enzymes. As an example, the substrate specificity of acloned β3GnT (reported values) is also shown in Table 4 [Proc. Natl.Acad. Sci. USA, 96, 406 (1999)].

By comparing Tables 2 and 4 with Table 6 which will be described later,it was confirmed also that, similar to the results of Example 14, theacetylglucosaminyltransferase (G3) is an enzyme having a substratespecificity which is clearly different from that of the other β1,3N-acetylglucosaminyltransferases (G4 and G7) obtained by the presentinvention. TABLE 4 Substrate specificity of aβ1,3-N-acetylglucosaminyltransferase (G3) with unlabeledoligosaccharides as substrates Relative activity (%) Substrate Sugarchain structure G3 β3GnT LNnT Galβ1-4GlcNAcβ1-3Galβ1-4Glc 100 100 LNTGalβ1-3GlcNAcβ1-3Galβ1-4Glc 27 6 LNnH Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-132 3Galβ1-4Glc Lactose Galβ1-4Glc 128 67.1 LacNAc Galβ1-4GlcNAc 21 95.5

On the other hand, when β1,3-galactosyltransferase activity of thesecreted G3 was measured by using GlcNAc and GlcNAcβ1-3Galβ1-4Glc asacceptor substrates, the activity was not detected. In consequence, itwas found that G3 does not have a β1,3-galactosyltransferase activity.The method described in Example 11(2) was used.

Example 16 Synthesis of a poly-N-acetyllactosamine Sugar Chain UsingSecreted β1,3-N-acetylglucosaminyltransferase (Secreted G3)

A poly-N-acetyllactosamine sugar chain was synthesized by using the FLAGpeptide-fused G3 polypeptide purified in Example 14 andβ1,4-galactosyltransferase.

(1) One-Pot Reaction

LNnT was allowed to react simultaneously with the FLAG peptide-fused G3polypeptide purified in Example 14 and β1,4-galactosyltransferase(manufactured by SIGMA) purified from bovine milk by the method shown inExample 12(2) to thereby synthesize a sugar chain in whichN-acetylglucosamine was added to the non-reducing end of LNnT(Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc) and a sugar chain inwhich a poly-N-acetyllactosamine sugar chain [Galβ1-4GlcNAcβ1-3)_(n) (nrepresents two or more)] was added to the non-reducing end of LNnT.

The specific reaction was carried out as follows.

The reaction was carried out at 37° C. for 5 hours in 30 μl of areaction solution [200 mmol/l MOPS (pH 7.5), 20 mmol/l UDP-GlcNAc(SIGMA), 50 mmol/l UDP-Gal (SIGMA), 20 mmol/l MnCl₂, 50 μmol/l of apyridylaminated sugar chain substrate, 10 μl of the G3 polypeptidepurified in Example 14, and 20 mU of β1,4-galactosyltransferase]. As thesubstrate, pyridylaminated LNnT was used, and formation of products wereconfirmed by HPLC. The method described in Example 11(1) was used.

As a result, a sugar chain in which [Galβ1-4GlcNAcβ1-3]_(n) (n=1, 2, 3or 4)] was added to the non-reducing end of pyridylaminated LNnT wassynthesized. In consequence, it was found that poly-N-acetyllactosaminesugar chains can be efficiently synthesized by a one-pot reaction byusing the G3 polypeptide and a β1,4-galactosyltransferase. It isconsidered that synthesis of longer poly-N-acetyllactosamine sugarchains is possible by increasing the amount of the enzymes, the amountof the substrate and the reaction time.

Example 17 Production of FLAG Peptide-Fusedβ1,3-N-acetylglucosaminyltransferase (G7) as a Secreted Form by UsingInsect Cell as a Host

Secreted expression of the FLAG peptide-fused G7 polypeptide in aninsect cell was carried out in the same manner as in Example 10.

(1) Construction of a Plasmid pVL1393-F2G7

It was considered based on its primary sequence that the G7 polypeptidecomprises an N-terminal cytoplasmic region containing 29 amino acids, asubsequent membrane-binding region rich in hydrophobic nature containing20 amino acids, a stem region containing at least 12 amino acids and theremaining C-terminal part comprising most of the polypeptide andcontaining a catalytic region. Accordingly, an attempt was made toeffect secreted expression of G7 polypeptide by removing the N-terminalcytoplasmic region containing 29 amino acids, the membrane-bindingregion containing 20 amino acids and a part of the stem region (6 aminoacids) and adding a immunoglobulin signal sequence and FLAG peptide tothe removed regions.

First, a plasmid pBlunt-G7 was constructed by preparing a DNA regionencoding a region considered to have the catalytic activity of G7polypeptide (from the 56th alanine to the 378th arginine in SEQ ID NO:4)by PCR and then inserting it into a vector pCR-Blunt vector. Thespecific method is described below.

A DNA shown by SEQ ID NO:33 (hereinafter referred to as “G7S-1”) and aDNA shown by SEQ ID NO:34 (hereinafter referred to as “G7S-2”) weresynthesized as primers for PCR.

They are designed such that a BglII site is generated in G7S-1, and aNotI site in G7S-2.

PCR was carried out by using Pyrobest DNA Polymerase manufactured byTaKaRa and 10× Pyrobest Buffer and 2.5 mmol/l dNTP Mixture attached tothe kit, in accordance with the instructions. Using a DNA thermal cycler(PERKIN ELMER CETUS DNA Thermal Cycler; manufactured by TaKaRa), 16cycles of a reaction, one cycle consisting of reaction at 94° C. for 30seconds, 65° C. for 1 minute and 72° C. for 2 minutes, were carried out,followed by reacting at 72° C. for 10 minutes. As the template, 20 ng ofa plasmid pT7B-G7 constructed in Example 4 was used. A DNA fragment ofabout 1.0 kb was obtained by the PCR. A plasmid pBlunt-G7 wasconstructed by inserting the DNA fragment into a vector pCR-Blunt. Theabsence of errors by the PCR was confirmed by determining nucleotidesequence of the DNA fragment inserted into pBlunt-G7.

By digesting pBlunt-G7 with restriction enzymes BglII and NotI, aBglII-NotI fragment of 1.0 kb encoding a region considered to have thecatalytic activity of G7 polypeptide (from the 56th alanine to the 378tharginine in SEQ ID NO:4) was obtained. By digesting pVL1393 withrestriction enzymes NotI and BstPI, a NotI-BstPI fragment of 6.4 kb wasobtained. By digesting a plasmid pVL1393-F2G4 constructed in Example 10with restriction enzymes BamHI and BstPI, a BamHI-BstPI fragment of 3.3kb was obtained pVL1393-F2G7 was constructed by linking these threefragments.

(2) Production of a Recombinant Virus

A recombinant virus was prepared for expression of the FLAGpeptide-fused G7 polypeptide as a secreted form in an insect cell. Arecombinant baculovirus was prepared by introducing a filamentousbaculovirus DNA and a plasmid pVL1393-F2G7 constructed in the above (1)into an insect cell Sf9 by a lipofectin method. The method described inExample 10 was used.

(3) Secreted Production and Purification of FLAG Peptide-Fused G7Polypeptide

Using the recombinant virus prepared in the above (2), a FLAGpeptide-fused G7 polypeptide was produced as a secreted form in aninsect cell. Thereafter, the polypeptide was purified from a culturesupernatant containing the polypeptide. The method described in Example10 was used.

SDS-PAGE was carried out by using 15 μl of a purified sample, and thenstaining was carried out by using Coomassie Brilliant Blue (FIG. 22).When a purified sample prepared from the culture supernatant of Sf21infected with the recombinant virus derived from pVL1393-F2G7 was used,a bands considered to be G7 polypeptide was found at a position ofranging from 40 to 42 kD. It is considered that difference in themolecular weight of each band is based on the number and size of theadded sugar chain. In the G7 polypeptide, there are threeN-glycosylation sites.

Based on the above result, it was shown that the FLAG peptide-fused G7polypeptide is produced as a secreted form in the insect cell culturesupernatant and can be purified easily by using Anti-FLAG M1 AffinityGel.

(4) Measurement of a β1,3-N-acetylglucosaminyltransferase Activity ofthe FLAG Peptide-Fused G7 Polypeptide

A β1,3-N-acetylglucosaminyltransferase activity of FLAG peptide-fused G7polypeptide was measured using 15 μl of the purified sample prepared inthe above (3). The method of Example 8 was used for the activitymeasurement. As a result, a β1,3-N-acetylglucosaminyltransferaseactivity was detected. The ratio of the substrate converted into theproduct was 2.1%. On the other hand, when the sample purified from theculture supernatant of Sf21 infected with a vector pVL1393 was used inthe same manner, the activity was not detected.

Based on the above result, it was shown that the FLAG peptide-fused G7polypeptide expressed as a secreted form in the insect cell has aβ1,3-N-acetylglucosaminyltransferase activity. The result showed that aβ1,3-N-acetylglucosaminyltransferase (G7) can be produced as a secretedform fused with the FLAG peptide in insect cells, and that sugar chainscan be synthesized by using the produced fusion protein.

Example 18 Examination of Substrate Specificity of the Secretedβ1,3-N-acetylglucosaminyltransferase (Secreted G7)

Examination of the substrate specificity of aβ1,3-N-acetylglucosaminyltransferase (G7) was carried out using the FLAGpeptide-fused G7 polypeptide purified in Example 17.

(1) Analysis Using Pyridylaminated Oligosaccharides as Substrates

Substrate specificity of the FLAG peptide-fused. G7 polypeptide wasexamined by the method shown in Example 11(1). The reaction was carriedout at 37° C. for 16 hours.

Relative activities when the activity for pyridylaminated LNnT used as asubstrate is defined as 100% are shown in Table 5. When LNnT was used asthe substrate, the conversion efficiency of the substrate into theproduct was 3.76%. It was found that theβ1,3-N-acetylglucosaminyltransferase (G7) uses LNnT having a type IIsugar chain (Galβ1-4GlcNAc) on the non-reducing end as a good substratebut hardly uses LNT or LNFP-V having a type I sugar chain(Galβ1-3GlcNAc) on the non-reducing end as a substrate. It was foundalso that an oligosaccharide LNFP-III in which fucose is added via anα1,3-linkage to the GlcNAc residue present in the second position fromthe non-reducing end of LNnT hardly becomes a substrate for G7. It wasfound also that oligosaccharides LNFP-II and LNDFH-II in which fucose isadded via an α1,4-linkage to the GlcNAc residue present in the secondposition from the non-reducing end of LNnT do not become substrates ofG7.

By comparing Table 1, Table 3 with Table 5, it was also found that theβ1,3-N-acetylglucosaminyltransferase (G7) is an enzyme having asubstrate specificity which is clearly different from that of the otherβ1,3-N-acetylglucosaminyltransferases (G3 and G4) obtained by thepresent invention. TABLE 5 Substrate specificity of aβ1,3-N-acetylglucosaminyltransferase (G7) with pyridylaminatedoligosaccharides as substrates Relative Substrate Sugar chain structureactivity (%) LNnT Galβ1-4GlcNAcβ1-3Galβ1-4Glc 100 LNFP-IIIGalβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc 7.4 LNT Galβ1-3GlcNAcβ1-3Galβ1-4Glc7.6 LNFP-II Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc 0 LNFP-VGalβ1-3GlcNAcβ1-3Galβ1-4(Fuc α1-3)Glc 8.1 LNDFH-IIGalβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1- 0 4(Fuc α1-3)Glc(2) Analysis by Using Unlabeled Oligosaccharides as Substrates

Substrate specificity of the FLAG peptide-fused G7 polypeptide wasexamined using the method shown in Example 11(2). The enzyme reactionwas carried out at 37° C. for 15.5 hours.

Relative activities when the activity for LNnT used as a substrate isdefined as 100% are shown in Table 6.

Conversion efficiency of the substrate into the product was 3.07% whenLNnT was used as a substrate. The β1,3-N-acetylglucosaminyltransferase(G7) used a tetraose LNnT as the best substrate. G7 used a disaccharidelactose as a relatively good substrate but hardly used a hexaose LNnH asa substrate. G7 also used a disaccharide LacNAc but the activity waslower than that for lactose. On the other hand, G7 did not use LNT as asubstrate.

Based on the above, it was considered that G7 can synthesizepoly-N-acetyllactosamine sugar chains of up to hexaose, but its activityof synthesizing poly-N-acetyllactosamine sugar chains of octaose or moreis considerably weak. It is known that already knownβ1,3-N-acetylglucosaminyltransferases use LacNAc as a good substraterather than lactose (J. Biol. Chem., 268, 27118 (1993), Proc. Natl.Acad. Sci. USA, 96, 406 (1999)]. In consequence, it was found that theβ1,3-N-acetylglucosaminyltransferase (G7) is an enzyme having asubstrate specificity which is clearly different from that of thealready known enzymes. As an example, substrate specificity of a clonedβ3GnT (reported values) is also shown in Table 6 [Proc. Natl. Acad. Sci.USA, 96, 406 (1999)].

By comparing Table 2, Table 4 with Table 6, it was confirmed also thatthe β1,3-N-acetylglucosaminyltransferase (G3) is an enzyme having asubstrate specificity which is clearly different from that of the otherβ1,3-N-acetylglucosaminyltransferases (G4 and G7) obtained according tothe present invention. TABLE 6 Substrate specificity of aβ1,3-N-acetylglucosaminyltransferase (G7) with unlabeledoligosaccharides as substrates Relative activity (%) Substrate Sugarchain structure G7 β3GnT LNnT Galβ1-4GlcNAcβ1-3Galβ1-4Glc 100 100 LNTGalβ1-3GlcNAcβ1-3Galβ1-4Glc 0 6 LNnH Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-0.05 3Galβ1-4Glc Lactose Galβ1-4Glc 32.6 67.1 LacNAc Galβ1-4GlcNAc 8.595.5

On the other hand, when β1,3-galactosyltransferase activity of thesecreted G7 was measured by using GlcNAc and GlcNAcβ1-3Galβ1-4Glc asacceptor substrates, the activity was not detected. In consequence, itwas found that G7 does not have a β1,3-galactosyltransferase activity.The method described in Example 11(2) was used.

Example 19 Examination of a Expression Level of Transcript of Each Genefor G3, G4 and G7 in Various Cancer Tissues

The expression level of transcript of each gene for G3, G4 and G7 invarious cancer tissues and normal tissues around the cancer tissues wasexamined. The examination was carried out in accordance with literatures[International Journal of Cancer, 83, 70 (1999), Glycobiology, 9, 607(1999), Laboratory Investigation, 78, 797 (1998)].

(1) Synthesis of Single-Stranded cDNAs Derived from Various NormalTissues and Cancer Tissues

Cancer tissues and normal tissues around the cancer tissues werecollected from patients of colon cancer (10 cases), gastric cancer (7cases) and lung cancer (6 cases). Total RNA was prepared from each ofthese tissues by the acid guanidium thiocyanate-phenol-chloroformmethod, and the synthesis of single-stranded cDNAs was carried out byusing the total RNAs as the template. Single-stranded cDNAs weresynthesized from 5 μg of the total RNA by using a kit (SUPERSCRIPTPreamplification System; manufactured by GIBCO), diluted 50 times withwater and used as the PCR templates. An Oligo(dT) primer was used as aprimer.

(2) Preparation of Standards and Internal Controls

The standards and internal controls were constructed by using pBS-G3,pBS-G4-2 and pT7B-G7 [cf. the following (a) to (f)].

In the determination of the amount of β-actin transcript, pUC119-ACT andpUC119-ACTd were converted into linear DNA fragments by digesting themwith restriction enzymes (HindIII and Asp718) capable of cutting out thecDNA moieties and used as a standard and an internal control,respectively [J. Biol. Chem., 269, 14730 (1994), Japanese PublishedUnexamined Patent Application No. 181759/94]. After confirming that eachplasmid was completely cut out, they were used by diluting stepwise,with water containing 1 μl/ml of yeast transfer RNA.

(a) Preparation of a Standard for Determination of the Amount of G3Transcript

A BglII fragment of 4.5 kb was obtained by digesting pBS-G3 with arestriction enzyme BglII. pBS-G3S was constructed by linking thisfragment. The G3 cDNA moiety was converted into linear DNA by digestingpBS-G3S with restriction enzymes XbaI and AccI, and used as a standardfor the determination. After confirming that the plasmid was completelycut out, it was used by diluting stepwise with water containing 1 μg/mlof yeast transfer RNA.

(b) Preparation of an Internal Control for Determination of G3 theAmount of Transcript

pBS-G3Sd was prepared by deleting 229 bp between Eco81I-PfIMI in the G3cDNA from pBS-G3S constructed in the above (a). An Eco81I-PfIMI-fragmentof 4.3 kb was obtained by digesting pBS-G3S with restriction enzymesEco81I and PfIMI. pBS-G3Sd was constructed by linking this fragment. TheG3 cDNA moiety was converted into linear DNA by digesting pBS-G3Sd withrestriction enzymes XbaI and AccI, and used as an internal control inthe determination. After confirming that the plasmid was completely cutout, it was used by diluting stepwise with water containing 1 μg/ml ofyeast transfer RNA.

(c) Preparation of a Standard for Determination of the Amount of G4Transcript

G4 cDNA moiety was converted into linear DNA by digesting pBS-G4-2obtained in Example 3 with restriction enzymes XbaI and ClaI, and usedas a standard for the determination. After confirming that the plasmidwas completely cut out, it was used by diluting stepwise with watercontaining 1 μg/ml yeast transfer RNA.

(d) Preparation of an Internal Control for Determination of the Amountof G4 Transcript

pBS-G4-2d was prepared by deleting 180 bp between BstEII-PmlI in the G4cDNA from the pBS-G4-2 obtained in Example 3. A BstEII-PmlI fragment of4.9 kb was obtained by digesting pBS-G4-2 with restriction enzymesBstEII and PmlI. pBS-G4-2d was constructed by linking this fragment. TheG4 cDNA moiety was converted into linear DNA by digesting the pBS-G4-2dwith XbaI and ClaI, and used as an internal control for thedetermination. After confirming that the plasmid was completely cut out,it was used by diluting stepwise with water containing 1 μq/ml of yeasttransfer RNA.

(e) Preparation of a Standard for Determination of the Amount of G7Transcription Product

G4 cDNA moiety was converted into linear DNA by digesting the pT7B-G7obtained in Example 4 with restriction enzymes Tth111I and NarI, andused as the standard for the determination. After confirming that theplasmid was completely cut out, it was used by diluting stepwise withwater containing 1 μl/ml yeast transfer RNA.

(f) Preparation of an Internal Control for Determination of the Amountof G7 Transcript

pT7B-G7d was prepared by deleting 208 bp between Tth111I-NarI in the G7cDNA from the pT7B-G7 obtained in Example 4. A Tth111I-NarI fragment of4.0 kb was obtained by digesting pT7B-G7 with restriction enzymesTth111I and NarI. pT7B-G7d was constructed by linking the fragment. TheG7 cDNA moiety was converted into linear DNA by digesting the pT7B-G7dwith HincII and SmaI, and used as the internal control for thedetermination. After confirming that the plasmid was completely cut out,it was used by diluting stepwise with water containing 1 μg/ml yeasttransfer RNA.

(3) Determination of the Amount of Transcript of Each Gene for G3, G4and G7 by the Quantitative PCR Method

PCR was carried out by using each single-stranded cDNAs prepared in theabove (1) from normal tissues and cancer tissues as templates. As PCRprimers, CB489 (SEQ ID NO:35) and CB490 (SEQ ID NO:36) were used fordetecting G3 transcript, and CB495 (SEQ ID NO:37) and CB523 (SEQ IDNO:38) for detecting G4 transcript, and CB493 (SEQ ID NO:39) and CB525(SEQ ID NO:40) for detecting G7 transcript. Also, calibration curveswere prepared by carrying out PCR in the same manner by using thestandards and internal controls prepared in the above (2) as respectivetemplates.

By using DNA polymerase AmpliTaqGold™ (manufactured by PERKIN ELMER),PCR was carried out in a reaction solution [10 mmol/l Tris-HCl (pH 8.3),50 mmol/l KCl, 1.5 mmol/l MgCl₂, 0.2 mmol/l dNTP, 0.001% (w/v) gelatinand 0.2 μmol/l each of gene-specific primers] containing 10 μl of cDNAderived from each of the above tissues and 10 μl (10 fg) of a plasmid asan internal control.

PCR was carried out under the following conditions.

In the determination of the amount of G3 transcript, the reactionsolution was heated at 95° C. for 11 minutes and then 42 cycles of areaction was carried out, one cycle consisting of reaction at 95° C. for1 minute, 55° C. for 1 minute and 72° C. for 2 minutes.

In the determination of the amount of G4 transcript, the reactionsolution was heated at 95° C. for 11 minutes and then 42 cycles of areaction was carried out, one cycle consisting of reaction at 95° C. for1 minute, 65° C. for 1 minute and 72° C. for 2 minutes.

In the determination of the amount of G7 transcript, the reactionsolution was heated at 95° C. for 11 minutes and then 44 cycles of areaction was carried out, one cycle consisting of reaction at 95° C. for1 minute, 65° C. for 1 minute and 72° C. for 2 minutes as one cycle.

In the determination of the amount of β-actin transcript, the reactionsolution was heated at 95° C. for 11 minutes and then 24 cycles of thereaction was carried out, one cycle consisting of reaction at 95° C. for1 minute, 65° C. for 1 minute and 72° C. for 2 minutes as one cycle.

A 10 μl portion of each solution after. PCR was subjected to 1% agarosegel electrophoresis, and the gel was stained with ethidium bromide andphotographed. By scanning the photograph by use of an NIH image system,stained intensity of amplified fragments were measured and used as theamount. In order to determine the amount of transcripts more accurately,similar PCR was carried out by changing the number of cycles of PCR.Amounts of the standards and internal controls were changed depending onthe number of cycles of the PCR.

By carrying out PCR using 1.25 fg, 2.5 fg, 5 fg, 10 fg, 20 fg and 40 fgof the standards prepared in the above (a), (c) and (e) instead of thecell-derived single-stranded cDNAs, amounts of amplified fragments weremeasured to prepare calibration curves by plotting the amount of cDNAand the amount of the amplified fragment.

When the primers for determination of the amount of G3 transcript areused, a DNA fragment of 647 bp is amplified from the G3 transcript andG3 standard; and a DNA fragment of 418 bp from the G3 internal control.

When the primers for determination of the amount of G4 transcriptionproduct are used, a DNA fragment of 498 bp is amplified from the G4transcription product and G4 standard, and a DNA fragment of 318 bp fromthe G4 internal control.

When the primers for determination of the amount of G7 transcriptionproduct are used, a DNA fragment of 619 bp is amplified from the G7transcription product and G7 standard, and a DNA fragment of 411 bp fromthe G7 internal control.

From the above calibration curve and the amount of amplified DNAfragments derived from each tissue, the amount of cDNA in each tissuewas calculated and used as the amount of the transcription product.Also, since β-actin is considered to be a gene universally expressed ineach tissue, it is considered that the expression level is almost thesame in every tissue. Accordingly, a difference in the expression levelof β-actin transcript in each tissue was considered to be the differencein the efficiency of cDNA synthesizing reaction, so that the expressionlevel of β-actin transcription product was also taken into considerationwhen the expression level of the respective genes were compared.

Amounts of the G3, G4 and G7 transcription products in cancer tissuesand peripheral normal tissues of colon dancer patients (10 cases) areshown in Table 7 as relative values when the amount of β-actintranscript is defined as 1,000.

Expression of G3 and G4 transcripts was found in cancer tissues andperipheral normal tissues of colon patients in most cases, but nocorrelation was found between the expression level in cancer tissues andnormal tissues. On the other hand, G7 transcript was hardly expressed inany of the cancer tissues and normal tissues. It can be considered thatthe expression hardly occurred when the expression (relative value) was1 or less. TABLE 7 Amount of Sample Patient transcription product nameNo. Tissue G3 G4 G7 10N 10 normal 35 27 0.51 10T 10 cancer 5.6 5.1 0.2011N 11 normal 2.3 5.1 0.17 11T 11 cancer 4.4 7.4 0.12 13N 13 normal 7.85.3 0.055 13T 13 cancer 6.0 0.070 0.01> 15N 15 normal 0.01> 0.01> 0.01>15T 15 cancer 5.4 5.0 0.01> 17N 17 normal 3.9 5.1 0.085 17T 17 cancer4.4 24 1.5 18N 18 normal 6.9 35 0.086 18T 18 cancer 3.3 5.6 0.01> 19N 19normal 7.4 6.3 0.01> 19T 19 cancer 3.8 6.4 0.16 22N 22 normal 3.6 4.00.01> 22T 22 cancer 8.6 5.0 0.01> 23N 23 normal 3.5 4.9 0.01> 23T 23cancer 4.6 5.2 0.057 24N 24 normal 4.9 7.3 0.14 24T 24 cancer 3.4 6.20.090

Amounts of the G3, G4 and G7 transcripts in cancer tissues andperipheral normal tissues of gastric cancer patients (7 cases) are shownin Table 8 as relative values when the amount of β-actin transcript isdefined as 1,000.

Expression of G3 and G4 transcripts was found in cancer tissues andperipheral normal tissues of gastric cancer patients in most cases, butno correlation was found between the expression level in cancer tissuesand normal tissues. On the other hand, G7 transcript was hardlyexpressed in any of the cancer tissues and normal tissues. TABLE 8Amount of Sample Patient transcription product name No. Tissue G3 G4 G7MK2N MK2 normal 56 120 0.01> MK2T MK2 cancer 8.5 12 0.26 MK4N MK4 normal3.2 14 0.067 MK4T MK4 cancer 4.3 4.8 0.038 MK5N MK5 normal 0.01> 4.50.059 MK5T MK5 cancer 4.6 6.0 0.26 MK6N MK6 normal 6.0 8.0 0.01> MK6TMK6 cancer 8.6 8.6 0.077 MK7N MK7 normal 12 12 0.01> MK7T MK7 cancer 1817 0.15 MK10N MK10 normal 7.3 5.5 0.01> MK10T MK10 cancer 5.8 4.0 0.18MK12N MK12 normal 4.8 12 0.01> MK12T MK12 cancer 17 13 0.01>

Amounts of the G3, G4 and G7 transcripts in cancer tissues andperipheral normal tissues of lung cancer patients (6 cases) are shown inTable 9 as relative values when the amount of β-actin transcript isdefined as 1,000.

Expression of G3 transcript was found in cancer tissues and peripheralnormal tissues of lung cancer patients in all cases, but no correlationwas found between the expression level in cancer tissues and normaltissues. G7 transcript was hardly expressed in any of the cancer tissuesand normal tissues.

Regarding G4 transcript, the expression was hardly found in normaltissues, while the significant expression was found in cancer tissues in5 cases among the 6 cases. The result suggests that G4 transcript isexpressed accompanied by the malignant transformation. TABLE 9 Amount ofSample Patient transcription product name No. Tissue G3 G4 G7 LC11N LC11normal 45 0.01> 0.37 LC11T LC11 cancer 4.2 1.5 0.082 LC12N LC12 normal7.9 0.01> 0.059 LC12T LC12 cancer 12 3.4 0.14 LC15N LC15 normal 8.60.01> 0.077 LC15T LC15 cancer 16 4.8 0.33 LC20N LC20 normal 27 0.20 0.25LC20T LC20 cancer 19 2.9 0.66 LC23N LC23 normal 3.2 0.01> 0.01> LC23TLC23 cancer 3.9 0.01> 0.12 LC25N LC25 normal 17 0.056 0.15 LC25T LC25cancer 5.3 2.2 0.16

Accordingly, the same analysis was carried out on other 16 cases of lungcancer patients. Including the results of above 6 cases, expressionlevel of G3, G4 and G7 transcripts in cancer tissues and peripheralnormal tissues of lung cancer patients (22 cases) are shown in Table 10as relative values when the amount of β-actin transcript is defined as1,000.

The expressed amounts are shown by arranging them according to theclassification of lung cancer.

Amounts of the G3, G4 and G7 transcripts in cancer tissues andperipheral normal tissues of lung cancer patients (22 cases) are shownas relative values when the amount of β-actin transcript is defined as1,000. TABLE 10 Amount of G4 Patient Classification transcriptionproduct No. of lung cancer Normal tissue Cancer tissue LC2Adenocarcinoma 0.01> 0.94 LC9 Adenocarcinoma 0.10 2.2 LC11Adenocarcinoma 0.01> 1.2 LC12 Adenocarcinoma 0.01> 2.3 LC13Adenocarcinoma 0.01> 9.8 LC15 Adenocarcinoma 0.14 2.9 LC17Adenocarcinoma 0.21 2.0 LC21 Adenocarcinoma 0.01> 6.4 LC24Adenocarcinoma 0.01> 0.01> LC25 Adenocarcinoma 0.01> 1.4 LC26Adenocarcinoma 0.01> 1.7 LC28 Adenocarcinoma 0.33 2.6 LC8 Adenocarcinoma(mod) 0.01> 5.8 LC14 Adenocarcinoma (mod) 1.0 7.6 LC10 Adenocarcinoma(well) 0.01> 1.5 LC18 Adenocarcinoma (well) 0.01> 2.5 LC3 Squamouse cellcarcinoma 0.018 0.56 LC6 Squamouse cell carcinoma 0.01> 0.21 LC16Squamouse cell carcinoma 0.027 3.4 LC20 Squamouse cell carcinoma 0.532.6 LC23 Mesothelioma 0.21 0.14 LC27 Small cell carcinoma 0.01> 0.11

When an expression level (relative value) of 1 or more is regarded as“expresssed”, expression of G4 transcript expressed in normal tissueswas seen in only one case among the total of 22 cases, and the expressedamount in this case was a low value of 1. On the other hand, G4transcript in cancer tissues was seen in 17 cases among the total of 22cases. Also, in the only one case in which expression of G4 transcriptwas found in normal tissues (LC14 in the table), the expression level incancer tissues was increased to 7.6 evidently accompanied by themalignant transformation. When only the cases of adenocarcinoma areconsidered, it can be seen that expression level of G4 transcript isincreased in 14 cases among the total of 15 cases (excluding LC24 in thedrawing) accompanied by the malignant transformation. In squamouse cellcarcinoma, correlation between malignant transformation and expressionlevel of G4 transcript was found in 2 cases among 4 cases.

The results show that G4 transcript is expressed accompanied by themalignant transformation in lung cancers (particularly inadenocarcinoma). In the only one case in which expression of G4transcript was found in normal tissues (LC14 in the drawing), there is apossibility that the normal tissue was contaminated with a cancertissue. Since the G4 gene is hardly expressed in normal lung tissues, itis considered that this is a gene which is expressed for the first timeaccompanied by the malignant transformation. In consequence, it isconsidered that lung cancers can be diagnosed by examining expressedamounts of the G4 gene and G4 protein in lung tissues.

INDUSTRIAL APPLICABILITY

The present invention can provide a novel polypeptide havingβ1,3-N-acetylglucosaminyltransferase activity; a method for producingthe polypeptide; a DNA which encodes the polypeptide; a recombinantvector into which the DNA is inserted; a transformant carring therecombinant vector; an antibody which recognizes the polypeptide; amethod for determining and immunologically staining the polypeptide ofthe present invention, using the antibody; a method for producing asugar chain having a GlcNAcβ1-3Gal structure, a poly-N-acetyllactosaminesugar chain and a complex carbohydrate containing the sugar chain, usingthe polypeptide; a method for producing a sugar chain having aGlcNAcβ1-3Gal structure, a poly-N-acetyllactosamine sugar chain and acomplex carbohydrate containing the sugar chain, by using thetransformant having the recombinant vector; a method for screening asubstance capable of changing the expression of a gene encoding thepolypeptide; a method for screening a substance capable of changing aβ1,3-N-acetylglucosaminyltransferase activity of the polypeptide; amethod for diagnosing inflammatory diseases and cancers (colon cancer,pancreatic cancer, gastric cancer and the like), by using the DNA or theantibody; and a method for treating inflammatory diseases and cancers(colon cancer, pancreatic cancer, gastric cancer and the like), by usingthe DNA, a substance capable of changing the expression of a geneencoding the polypeptide or a substance capable of changing theβ1,3-N-acetylglucosaminyltransferase activity of the polypeptide.

Free Text of Sequence Listing

-   SEQ ID NO:8—Nucleotide sequence of G7 cNDA-   SEQ ID NO:9—Explanation of synthetic sequence: Synthetic DNA-   SEQ ID NO:10—Explanation of synthetic sequence: Synthetic DNA-   SEQ ID NO:11—Explanation of synthetic sequence: Synthetic DNA-   SEQ ID NO:12—Explanation of synthetic sequence: Synthetic DNA-   SEQ ID NO:13—Explanation of synthetic sequence: Synthetic DNA-   SEQ ID NO:14—Explanation of synthetic sequence: Synthetic DNA-   SEQ ID NO:15—Explanation of synthetic sequence: Synthetic DNA-   SEQ ID NO:16—Explanation of synthetic sequence: Synthetic DNA-   SEQ ID NO:17—Explanation of synthetic sequence: Amino acid sequence    of FLAG peptide-   SEQ ID NO:18—Explanation of synthetic sequence: Synthetic DNA-   SEQ ID NO:19—Explanation of synthetic sequence: Synthetic DNA-   SEQ ID NO:20—Explanation of synthetic sequence: Synthetic DNA-   SEQ ID NO:21—Explanation of synthetic sequence: Synthetic DNA-   SEQ ID NO:22—Explanation of synthetic sequence: Synthetic DNA-   SEQ ID NO:23—Explanation of synthetic sequence: Synthetic DNA-   SEQ ID NO:24—Explanation of synthetic sequence: Synthetic DNA-   SEQ ID NO:25—Explanation of synthetic sequence: Synthetic DNA-   SEQ ID NO:26—Explanation of synthetic sequence: Synthetic DNA-   SEQ ID NO:27—Explanation of synthetic sequence: Synthetic DNA-   SEQ ID NO:28—Explanation of synthetic sequence: Synthetic DNA-   SEQ ID NO:29—Explanation of synthetic sequence: Synthetic DNA-   SEQ ID NO:30—Explanation of synthetic sequence: Synthetic DNA-   SEQ ID NO:31—Explanation of synthetic sequence: Synthetic. DNA-   SEQ ID NO:32—Explanation of synthetic sequence: Synthetic DNA-   SEQ ID NO:33—Explanation of synthetic sequence: Synthetic DNA-   SEQ ID NO:34—Explanation of synthetic sequence: Synthetic DNA-   SEQ ID NO:35 Explanation of synthetic sequence: Synthetic DNA-   SEQ ID NO:36—Explanation of synthetic sequence: Synthetic DNA-   SEQ ID NO:37—Explanation of synthetic sequence: Synthetic DNA-   SEQ ID NO:38—Explanation of synthetic sequence: Synthetic DNA-   SEQ ID NO:39—Explanation of synthetic sequence: Synthetic DNA-   SEQ ID NO:40—Explanation of synthetic sequence: Synthetic DNA

1-33. (canceled)
 34. A process for producing a sugar chain or complexcarbohydrate, which comprises: culturing a transformant carrying arecombinant DNA obtained by inserting a DNA encoding a polypeptide whichis the active ingredient of a sugar chain synthesizing agent comprisingthe amino acid sequence represented by SEQ ID NO:2 into a vector in amedium to produce and accumulate a sugar chain comprising a saccharideselected from the group consisting of a saccharide having aGlcNAcβ1-3Gal 1-4GlcNAc structure, a saccharide having aGlcNAcβ1-3Galβ1-3GlcNAc structure, a saccharide having aGlcNAcβ1-3Galβ1-4Glc structure, a saccharide having a(Galβ1-4GlcNAcβ1-3)_(n)Galβ1-4GlcNAc structure wherein n is 1 or more,and a saccharide having a (Galβ1-4GlcNAcβ1-3)_(n)Galβ1-4Glc structurewherein n is 1 or more, or a complex carbohydrate comprising the sugarchain, in the culture; and recovering the sugar chain or complexcarbohydrate from the culture.
 35. The process according to claim 34,wherein the transformant is a microorganism, or an animal cell. 36-62.(canceled)
 63. A process for producing a sugar chain or complexcarbohydrate, which comprises: culturing a transformant carrying arecombinant DNA obtained by inserting a DNA encoding a polypeptide whichis the active ingredient of a sugar chain synthesizing agent comprisingthe amino acid sequence positions 45-372 represented by SEQ ID NO:2 intoa vector in a medium to produce and accumulate a sugar chain comprisinga saccharide selected from the group consisting of a saccharide having aGlcNAcβ1-3Galβ1-4GlcNAc structure, a saccharide having aGlcNAcβ1-3Galβ1-3GlcNAc structure, a saccharide having aGlcNAcβ1-3Galβ1-4Glc structure, a saccharide having a(Galβ1-4GlcNAcβ1-3)_(n)Galβ1-4GlcNAc structure wherein n is 1 or more,and a saccharide having a (Galβ1-4GlcNAcβ1-3)_(n)Galβ1-4Glc structurewherein n is 1 or more, or a complex carbohydrate comprising the sugarchain, in the culture; and recovering the sugar chain or complexcarbohydrate from the culture.
 64. The process according to claim 63,wherein the transformant is a microorganism, or an animal cell.
 65. Aprocess for producing a sugar chain or complex carbohydrate, whichcomprises: culturing a transformant carrying a recombinant DNA obtainedby inserting a DNA encoding a polypeptide which is the active ingredientof a sugar chain synthesizing agent comprising the amino acid sequencerepresented by SEQ ID NO:3 into a vector in a medium to produce andaccumulate a sugar chain comprising a saccharide selected from the groupconsisting of a saccharide having a GlcNAcβ1-3Galβ1-4GlcNAc structure, asaccharide having a GlcNAcβ1-3Galβ1-3GlcNAc structure, a saccharidehaving a GlcNAcβ1-3Galβ1-4Glc structure, a saccharide having a(Galβ1-4GlcNAcβ1-3)_(n)Galβ1-4GlcNAc structure wherein n is 1 or more,and a saccharide having a (Galβ1-4GlcNAcβ1-3)_(n)Galβ1-4Glc structurewherein n is 1 or more, or a complex carbohydrate comprising the sugarchain, in the culture; and recovering the sugar chain or complexcarbohydrate from the culture.
 66. The process according to claim 65,wherein the transformant is a microorganism, or an animal cell.
 67. Aprocess for producing a sugar chain or complex carbohydrate, whichcomprises: culturing a transformant carrying a recombinant DNA obtainedby inserting a DNA encoding a polypeptide which is the active ingredientof a sugar chain synthesizing agent comprising the amino acid sequencepositions 45-372 represented by SEQ ID NO:3 into a vector in a medium toproduce and accumulate a sugar chain comprising a saccharide selectedfrom the group consisting of a saccharide having aGlcNAcβ1-3Galβ1-4GlcNAc structure, a saccharide having aGlcNAcβ1-3Galβ1-3GlcNAc structure, a saccharide having aGlcNAcβ1-3Galβ1-4Glc structure, a saccharide having a(Galβ1-4GlcNAcβ1-3)_(n)Galβ1-4GlcNAc structure wherein n is 1 or more,and a saccharide having a (Galβ1-4GlcNAcβ1-3)_(n)Galβ1-4Glc structurewherein n is 1 or more, or a complex carbohydrate comprising the sugarchain, in the culture; and recovering the sugar chain or complexcarbohydrate from the culture.
 68. The process according to claim 67,wherein the transformant is a microorganism, or an animal cell.